Methods and compositions using cellular asialodeterminants and glycoconjugates for targeting cells to tissues and organs

ABSTRACT

The present invention is directed to methods for delivering cells to a target tissue in a mammal using glycoconjugate to traffic the cell to a desired organ in the mammal. The methods according to the present invention are especially applicable to administering lymphoid cells such as natural killer (NK) cells activated with interleukin-2 (IL-2), lymphokine-activated killer (LAK) cells and/or tumor-infiltrating lymphocytes (TILs) and/or cytotoxic lymphocytes (CTLs), or stem cells such as those derived from the bone marrow or from umbilical cord tissue. The methods are also useful for targeting a gene of interest to a tissue in a mammal by introducing a cell containing the gene of interest and administering a glycoconjugate to the mammal.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/364,498, filed Mar. 15, 2002, the entirety of whichis incorporated by reference herein for all purposes.

FIELD OF THE INVENTION

The present invention is in the field of clinical medicine and therapy.The invention relates to methods and compositions for targeting cells toan organ of interest, using sialo- or asialodeterminants, particularlyneoasialodeterminants, on cell surfaces and/or on free glycoconjugates.

BACKGROUND OF THE INVENTION

Morell et al. determined that when a sialyl group of ceruloplasmin isremoved by neuraminidase, this plasma protein rapidly disappears fromserum. They disclosed that this phenomenon is due to the uptake by theasialoglycoprotein (ASGP) receptor present in liver cells (J. Biol.Chem., 243:155 (1968)). Thereafter, it was reported that the ASGPreceptor is present only in liver cells (Adv. Enzymol., 41:99, (1974)).Such specific uptake by liver cells has been identified from the factthat when asialoceruloplasmin or asialoorosomucoid, which isexperimentally labeled with tritium, is injected into the living body,the isotope is selectively detected only in liver cells. Scheinberg, I.H., et al., Hepatic removal of circulating proteins, in Davidson C. S.,ed. Problems in Liver Diseases, pp. 279-285, New York, Stratton Company,(1979). In addition, it was also disclosed that this receptorspecifically recognizes and absorbs glycoproteins having D-galactose orN-acetylgalactosamine as the terminal sugar group (Ann. Rev. Biochem.51:531, (1982)).

The cell membrane of liver cells comprises a cell structure whichcombines with asialoglycoprotein terminated with galactose. This cellstructure was first named hepato-binding protein (HBP) but is presentlycalled asialoglycoprotein (ASGP) receptor. Further, it has been observedthat among various desialylated glycoproteins, the desialylatedalpha(1)-acid glycoprotein, asialoorosomucoid, most rapidly disappearsfrom the serum after injection. Therefore, it has been determined thatasialo-alpha(1)-acid glycoprotein is both specifically and well taken upby liver cells (J. Biol. Chem., 245:4397 (1970)). The ASGP receptor isconstituted with a single polypeptide having a molecular weight of about40,000 and can recognize a glycoprotein having a galactose residue atthe nonreductive terminal position of the saccharide chain (i.e.,asialoglycoprotein).

While the physiological functions of an ASGP receptor are stilluncertain, it is believed that an ASGP receptor participates in themetabolism of glycoproteins. In fact, the increase of the blood level ofan ASGP is observed in case of hepatic diseases such as chronichepatitis, liver cirrhosis and hepatic cancer. Further, the decrease ofthe quantity of an ASGP receptor is observed in an experimental model ofhepatic disorder induced by administration of chemicals.

In view of these phenomena, it may be possible to diagnose hepaticdiseases through assessment of the quantity and quality of an ASGPreceptor determined by the use of an ASGP-like substance, i.e., an ASGPreceptor-directing compound. In fact, asialoglycoconjugates have beencovalently linked to other agents as a means of targeting chemical(immunosuppressive drugs) and biological agents (antibodies) to be takenup by the liver for therapeutic and diagnostic purposes (see, e.g., U.S.Pat. Nos. 5,346,696, 5,679,323, and 5,089,604).

Adoptive cellular immunotherapy in general is a treatment that employsbiological reagents to effect an immune-mediated response. Currently,most adoptive immunotherapies are autolymphocyte therapies (ALT)directed to treatments using the patient's own immune cells which havebeen processed to either enhance the immune cell mediated response or torecognize specific antigens or foreign substances in the body, includingcancer cells. The treatments are accomplished by removing the patient'slymphocytes and exposing these cells in vitro to biologics and drugs toactivate the immune function of the cells. Once the autologous cells areactivated, these ex vivo activated cells are reinfused into the patientto enhance the immune system to treat various forms of cancer,infectious diseases, autoimmune diseases or immune deficiency diseases.

Adoptive immunotherapies may utilize, for instance, natural killer (NK)cells activated with interleukin-2 (IL-2), lymphokine-activated killer(LAK) cells and/or tumor-infiltrating lymphocytes (TILs) and/orcytotoxic lymphocytes (CTLs). LAK therapy involves the in vitrogeneration of LAK cells by culturing autologous peripheral bloodleukocytes in high concentrations of IL-2. The LAK cells are thenreinfused into the cancer patient in a treatment that may also involvesinfusion of IL-2. Rosenberg, et al., “Cancer immunotherapy usinginterleukin-2 and interleukin-2 activated lymphocytes,” Annual Review ofImmunology 4:681-709 (1986). TIL therapy involves the generation of LAKcells from mononuclear cells originally derived from the inflammatoryinfiltrating cells present in and around solid tumors, obtained fromsurgical resection specimens. Rosenberg, et al., “A new approach to theadoptive immunotherapy of cancer with tumor-infiltrating lymphocytes,”Science 233:1318-1321 (1986). Many further variations of adoptiveimmunotherapy have been developed in recent years. See, e.g., U.S. Pat.No. 6,406,699, issued Jun. 18, 2002 to Wood, disclosing and claiming acomposition and method of cancer antigen immunotherapy, and methods inreferences disclosed and cited therein.

In addition to cancer immunotherapies, adoptive immunotherapy hasapplications for deficiency or dysfunction of T cells associated withseveral diseases and conditions, including recurrent infections byviruses such as herpesvirus (HSV, VZV, CMV), hepatitis B virus, andpapillomavirus. See, e.g., Spiegel, R. J., “The alpha interferons:Clinical overview”, Seminars in Oncology 14:1 (1987). ALT is also beingevaluated in the treatment of patients infected with HIV. O.Martinez-Maza, “HIV-Induced Immune Dysfunction and AIDS-AssociatedNeoplasms,” in Biological Approaches to Cancer Treatment: Biomodulation,M. Mitchell, Editor, McGraw-Hill, Inc., Chapter 9, pages 181-204 (1993).

A stem cell is a special kind of cell that has a unique capacity torenew itself and to give rise to specialized cell types. Although mostcells of the body such as heart cells or skin cells, are committed toconduct a specific function, a stem cell is uncommitted and remainsuncommitted until it receives a signal to develop into a specializedcell. In 1998, stem cells from early human embryos were first isolatedand grown in culture. It is recognized that these stem cells are,indeed, capable of becoming almost all of the specialized cells of thebody. In recent years, stem cells present in adults also have been shownto have the potential to generate replacement cells for a broad array oftissues and organs, such as the heart, the liver, the pancreas, and thenervous system. Thus, this class of adult human stem cell holds thepromise of being able to repair or replace cells or tissues that aredamaged or destroyed by many devastating diseases and disabilities. Itis highly useful to effect such therapies by targeting stem cells toparticular organs of the body.

In the prior art, lymphocytes and stem cells generally have beenpresented to the desired organs either by injection into the tissue orby infusion into the local circulation. However, localization of normalbone marrow stem cells and lymphocytes to the liver has beendemonstrated upon injection of such cells into mice. Samlowski et al.,Immunol. 88:309-322 (1984); Samlowski et al., Proc. Natl. Acad. Sci.82:2508-2512 (1985).

It is also known that a large proportion of cells infused into mammalsadhere to the lung endothelium, independent of cell type orphysiological homing properties. It has been observed that stem cellsaccumulate in the lungs when they are administered. Morrison et al.,Nature Medicine 2:1281-1282 (1996); Martino et al., Eur. J. Immunol.23:1023-1028 (1993); Pereira et al., Proc. Natl. Acad. Sci. USA92:4857-4861 (1993); and Gao et al., Cells Tissues Organs 169:12-20(2001).

Orosomucoid, asialo-orosomucoid and agalacto/asialo-orosomucoid havebeen shown to inhibit neutrophil activation, superoxide aniongeneration, and platelet activation. Costello et al., Clin Exp Immunol55:465-472 (1984); and Costello et al., Nature 281:677-678 (1979). Theseproteins also induced transient immunosuppression and protected againstTNF challenge. Bennett, et al., Proc. Natl. Acad. Sci. USA 77:6109-6113(1980) and Libert, et al., J. Exp. Med. 180:1571-1575 (1994).Orosomucoid demonstrated specific binding to pulmonary endothelialcells, which appeared to be independent of carbohydrate recognitionsites. Schnitzer, et al., Am. J. Physiol 263:H48-H55 (1992). Moreover,orosomucoid was shown to bind to skin capillary endothelial cells in adose dependent manner, thereby maintaining normal capillary permeabilityin the face of inflammatory agonists that caused leakage in controlanimals. Muchitsch, et al., Arch Int Pharmacodyn 331:313-321 (1996).Similarly, infused orosomucoid bound to kidney capillaries and restoredthe permselectivity of glomerular filtration. Muchitsch, et al., Nephron81:194-199 (1999).

Entrapment of neuraminidase-treated lymphocytes in the liver also hasbeen reported, including autoimmune reactions against liver cells bysyngeneic neuraminidase-treated lymphocytes, in mice intravenouslyinjected with lymphocytes isolated from spleen or thymus. Kolb-Bachofen,V., et al., Immunol. 123:2830-2834 (1979). Studies on interactionsbetween neuraminidase-treated rat lymphocytes and liver cells in culturehave demonstrated adhesion between cells is due to stereo-specificinteractions between a mammalian hepatic membrane lectin (i.e., the ASGPreceptor) and galactosyl residues which are exposed on the lymphocytesurface after removal of sialic acid residues. Kolb, H., et al., Adv.Exp. Med. Biol. 114:219-222 (1979).

In view of the above, a need exists to develop methods for delivery oflymphocytes and stem cells through the circulation to specific organs.Such methods would provide a means to target non-invasively solid organssuch as the liver, heart, lungs and kidneys. In addition, very diffusetissues, such as the lung, which are not amenable to dosage by injectioncould be targeted. Such methods would be useful in adoptiveimmunotherapies and regenerative stem cell therapies involving suchorgans as the liver, heart, lungs and kidneys.

The present invention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention features a method for delivering a cell to atarget tissue in a mammal comprising the steps of administering acarbohydrate presenting molecule (e.g., a glycoconjugate) to a mammaland then administering the cell to the mammal.

As used herein, the term “administering” refers to any method ofinducing an increased concentration of the cell in the circulation ofthe mammal, whether by infusion from an extraneous source or bymobilizing the cell into the circulation from a depot within the mammal,such as the marrow. Means for mobilizing stem cells, for instance, usingGM-CSF and GCSF, for example, are well known in the art. See, e.g.,Simmons et al., The mobilization of primitive hemopoietic progenitorsinto the peripheral blood. Stem Cells, 12 Suppl 1:187-201 (1994).

The methods according to the present invention are especially applicableto stem cells, such as those derived from the bone marrow, peripheralblood, umbilical cord or from mesenchymal stem cells expanded inculture. The stem cells within the scope of the invention include anycell capable of differentiating into a desired target tissue. Such cellsinclude pluripotent stem cells, embryonic stem cells, multipotent adultstem cells, and progenitor or precursor cells.

The methods according to the present invention also are especiallyapplicable to immune system cells, such as natural killer (NK) cellsactivated with interleukin-2 (IL-2), lymphokine-activated killer (LAK)cells and/or activated lymphocytes including but not limited totumor-infiltrating lymphocytes (TILs).

The methods of the present invention allow cells such as normal stem orimmune cells to be targeted to such target tissues as the heart, theliver, the kidneys and the lungs, among others. In some embodimentswherein the cell is targeted to the heart, the methods featureadministering an orosomucoid (O) or administering an asialoorosomucoid(ASO), and administering the cell to the mammal. In embodiments whereinthe cell is targeted to the lungs, the methods feature administering thecell to the mammal in a saline or a serum albumin-saline solution orcell culture media without protein/albumin. In embodiments wherein thecell is targeted to the liver, the methods feature administering anorosomucoid or an asialoorosomucoid and administering the cell to themammal. In some embodiments, the orosomucoid is administeredconcurrently or prior to administering the cell to the mammal. Themethods according to the present invention are also useful for eitherinhibiting or enhancing sequestration of a stem cell or immune cell inthe liver of a mammal even in the absence of targeting the cell to atarget organ.

The glycoconjugates of the present invention may be generallyrepresented by the general formula P-(S)x-Gal wherein P is a peptideresidue of a human serum glycoprotein and S is a sugar residue of ahuman serum glycoprotein; x is an integer from 1 to 100 and Gal isgalactose residue. The glycoconjugates may be partially or completelyasialylated. Especially useful glycoconjugates include fetuins,asialofetuins, orosomucoids and asialoorosomucoids.

The glycoconjugates may be administered to the mammal in any time framerelative to administering the cell. They may be administered before,after or simultaneously with the administration of the cell. In atypical embodiment, the glycoconjugates are administered prior to thecell. The glycoconjugates and the cell may be administered via anysuitable route. In preferred embodiments, they are administeredparenterally, and more preferably, intravenously to the mammal.

The methods according to the present invention are also useful fortargeting a gene of interest to a tissue in a mammal by introducing acell naturally containing, or a cell transformed with, the gene ofinterest to the mammal. Such methods are useful for treating a diseasecharacterized by a deficiency in a gene product in a mammal byadministering a cell comprising a functional gene encoding the geneproduct into the mammal and administering a glycoconjugate to themammal. According to these methods, a cell containing an exogenousfunctional gene of interest may be administered and localized to aparticular organ in the body where it can function to produce adeficient gene product.

Also, the methods according to the present invention are useful fortreating a disease characterized by tissue damage in a mammal byadministering a cell and administering a glycoconjugate to the mammal.Because stem cells have the potential to generate replacement cells fora broad array of tissues and organs, such as the heart, the pancreas,and the nervous system, stem cells may be targeted to particular organsin the body to repair or replace cells or tissues that are damaged ordestroyed by many devastating diseases and disabilities. In someembodiments, the disease may be a heart disease, a lung disease, akidney disease or a liver disease, for example, myocardial infarction,emphysema, cystic fibrosis, microalbuminuria, nephritis, stroke orhepatitis.

The methods according to the present invention are also useful fortreating a disease characterized by tissue damage in a mammal byadministering a glycoconjugate to the mammal and administering chemicalsor biopharmaceuticals that mobilize stem cells into the circulation. Theconcentration of circulating mobilized stem cells may be limited becausecertain organs may sequester stem cells, thereby limiting delivery of aneffective dose to the damaged organ. By inhibiting sequestration, theglycoconjugates of the invention increase the cell dose at the organ;thereby increasing the potential to generate replacement cells. Themethods including agents to mobilize stem cells also can be used for abroad array of tissues and organs, such as the heart, the pancreas, andthe nervous system. Mobilized stem cells may be targeted to particularorgans in the body to repair or replace cells or tissues that aredamaged or destroyed by many devastating diseases and disabilities. Insome embodiments wherein stem cells are mobilized, the disease may be aheart disease, a lung disease, a kidney disease, a neurological diseaseor a liver disease such as, for example, myocardial infarction,emphysema, cystic fibrosis, microalbuminuria, nephritis, stroke orhepatitis.

In other embodiments, the present invention provides pharmaceuticalcompositions comprising a cell and a glycoconjugate, e.g.; glycoprotein.Glycoproteins useful in the present invention include, for example,fetuins, orosomucoids (O) and asialoorosomucoids (ASO). In otheraspects, the present invention features an article of manufacture,comprising packaging material and a pharmaceutical agent containedwithin the packaging material, wherein the pharmaceutical agentcomprises a glycoconjugate of the invention that is therapeuticallyeffective for targeting a cell to a desired organ according to thepresent invention, and wherein the packaging material comprises a labelwhich indicates that the pharmaceutical agent can be used for targetinga cell to a desired organ according to the present invention. In someembodiments, the article of manufacture further comprises additionalreagents, such as solutions for making cell suspensions to beadministered, and/or printed instructions, for use in targeting cellsaccording to the invention. Such articles include, for instance, kitsfor treating tissue damage or for delivering a functional gene or geneproduct to a tissue in a mammal comprising a cell and a glycoprotein.Glycoproteins useful in the articles of manufacture of the inventioninclude fetuins, asialofetuins, orosomucoids and asialoorosomucoids.

In still other embodiments, the present invention provides methods forderivatization of stem cell or lymphoid cell populations to generate anasialodeterminant-bearing cell preparation to facilitate hepaticentrapment. In particular, the invention provides derivatized, activatedstem cells or lymphocytes that have asialadeterminants on their surfacethat have been generated by enzymatic or chemical means so that thesecells, when administered parenterally, circulate, are bound, andsequestered or entrapped by the liver via the ASGP receptor. Methods fortreating whole viable cells with a sialidase, such as neuraminidase, areknown in the art. See, e.g., Neubauer, R. H. et al., Identification ofnormal and transformed lymphocyte subsets of nonhuman primates withmonoclonal antibodies to human lymphocytes. J. Immunol. 130:1323-1329(1983); Kolb-Bachofen, V., et al., 1979, supra; and Kolb, H., et al.,1979, supra.

For instance, the invention provides a process of derivatization of stemcells to generate neoasialadourminants on the surface of such calls, forthe purpose of directing these tells to the liver to repair orregenerate liver functions and structures, or for delivery of normalgenes or genetically engineered cells for the purpose of curing orameliorating disease states. Operative elements of this aspect of theinvention are the ability to direct the localization of the transfusedstem cells bearing artificially created neoasialoglycodeterminants,their ability to create a micro-chimera of the recipient, and themechanism by which of the neodeterminants are specifically sequesteredby the liver. Thus, assimilation of theseneoasialoglycodeterminant-bearing cells would result in microchimerism(a mixture of derivatized stem cells and the original host cells thatwere genetically abnormal. The modified stem cells would express atleast the minimum required amount of the abnormal or missing protein orregulatory function needed for reversing or ameliorating the diseasephenotype. The modified stem cells could be derived from patient'sblood, bone marrow or other stem cell-producing organ such as adiposetissue, or may be derived from another individual or a stem cell line.

The invention also provides methods for manipulation of in vivo celltrafficking patterns of lymphoid cytolytic cells by specificallyfacilitating the hepatic sequestration of parenterally administeredactivated lymphocytes by derivitizing the cell surface with enzymes thatgenerate “neoasialodeterminants”. Thus, activated lymphoid populationshave cell surface asialodeterminants capable of binding to the ASGPreceptor, and this binding can be further enhanced by enzymatictreatments that generate new cell surface asialodeterminants.

These methods may be used, for instance, to improve the efficiency ofadoptive immunotherapy for liver metastasis or primary liver tumors byfacilitating hepatic entrapment (via the ASGP receptor) of parenterallyadministered cells that have been derivatized to generate cell surfaceasialodeterminants. Metastasis of various cancers to the liver aredifficult to treat. For example, elimination of breast cancer metastasesto liver must be achieved prior to harvesting of bone marrow orautologous stem cell products for transplantation. Chemotherapy alonecan take months to achieve a complete response. It often leads to bonemarrow suppression making the harvesting of stem cells from individualsextremely difficult. In cases where the tumor is chemotherapy resistant,very few therapeutic options remain. Adoptive immunotherapies do existin which the patients own cells can be “educated” in culture torecognize the tumor and then these cells are transferred to the patientintravenously to find and destroy the tumor.

If the tumor burden is primarily in the liver, it may be useful to haveseveral cycles of therapy directed specifically toward the eliminationof tumor from the liver. This can be accomplished according to thepresent invention, by treating the activated lymphoid populations (thathave been grown or “educated” in culture) with enzymes or othertreatments that include (but are not limited to sialidases, such asneuraminidases, that modify the cell surface glycosylation sites toexpose asialodeterminants. The number of these determinants are therebydramatically increased and hence the modified cells bind to hepatic ASGPreceptors more readily and dissociate less frequently than cells bearingthe “normal” number of asialodeterminants.

Assimilation of neoasialoglycodeterminant-bearing lymphoid cells resultsin microchimerism, as described for stem cells above, a mixture ofinfused lymphocytes that have or have not been genetically engineeredwith the original host lymphoid cells that exist at the site. Theinfused lymphoid cells would augment or enhance the immune response bydividing and entering the circulation and recruiting other cellpopulations to participate in the local immune response. The hepaticenvironment is ideally suited for the development of immune responsesdue to the presence of cells of the innate immune system as well asprofessional antigen presenting cells in the sinusoids and vasculature,particularly the portal system.

For example, several studies have shown that responses to metastaticcutaneous melanoma, for instance, can be achieved using regionaladministration to the liver of activated lymphocytes. See, e.g.,Keilhoiz, U. et al., Regional adoptive imunotherapy with interleukin-2and lymphokine-activated killer (LAK) cells for liver metastasis, Eur.J. Cancer 3OA:103-105 (1994). The invention methods of using activatedlymphocytes that have been modified to generate additional cell surfaceasialodeterminants permits the delivery of activated lymphocytes to theliver regionally, via the hepatic artery or portal vein or peripheralvein, without the use of invasive procedures to deliver these cells to aprimary hepatic or non-hepatic tumor, or to metastatic lesions distantfrom a primary hepatic or non-hepatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of liver entrapment of bone marrow stemcells and lymphocytes in the liver. Asialoglycodeterminants on thesurface of cells react with ASGP receptors on the surface of hepatocytesresulting in the localization of the bone marrow stem cells and thelymphocytes in the liver. Glycoconjugates includingasialoglycoconjugates block such interactions betweenasialoglycodeterminants on the surface of cells with ASGP receptors onthe surface of hepatocytes.

FIG. 2 shows the carbohydrate structure on two exemplary glycoproteinsof the invention.

FIG. 3 shows the relative binding affinities of different carbohydratesfor the ASGP receptor.

FIG. 4 shows the relative binding affinities of different carbohydratesfor the ASGP receptor.

FIG. 5 shows a schematic of an experimental system for studyingadherence of NK/LAK cells to monolayer cultures of (1) a human hepatomacell line (HEP2G), an asialoglyprotein repetor positive (ASGPR+) cellline that exhibits minimal deviation from cells in human liver tissue,and (2) a human renal cell carcinoma cell line (CAKI-2), an ASGPR− cellline.

FIG. 6 shows a plot of results of testing effects of asialofetuin (ASF)and fetuin (F) on adherence of NK/LAK cells (as represented by NK/LAKactivity) to HEP2G monolayers at 4° C. LAK activity (50%) adheres tohuman minimal deviation hepatoma, HEPG2, at 4° C., in the presence ofthe control fully sialylated protein, fetuin, F (LAK-NA/F). LAK activitydoes not adhere to the HEPG2 monolayer in the presence of asialofetuin,ASF (LAK-NA/ASF). LAK cells were incubated in the presence of ASF alone,i.e., no adherence to monolayer not performed (LAK/ASF). CONTROL cellsdid not kill RAJI targets (CONTROL). **This is representative of threedifferent donors.

FIG. 7 shows results of testing effects of asialofetuin (ASF) and fetuin(F) on adherence of NK/LAK cells to HEP2G and CAKI-2 cells at 23° C. Theeffector cell populations were: an untreated 3-day old LAK preparation(LAK) and the same population treated with Vibrio cholera neuraminidase(LAK/NS). LAK adherence to HEPG2 (ASGPR+) and CAKI-2 (ASGPR−) in thepresence of either ASF or F assayed on K562. (LAK=3 day LAK; K5=K562targets; FET=fetuin; ASF=asialofetujn; LAK/CAKI/ASF/K5=LAK, adherence onCAKI pretreated with ASF, assayed on K562).

FIG. 8 shows additional results of testing effects of asialofetuin (ASF)and fetuin (F) on adherence of NK/LAK cells to HEP2G and CAKI-2 cells at23° C., as in FIG. 7. Adherence of neuraminidase-treated LAK to HEPG2(ASGPR+) and CAKI-2 (ASGPR−) in the presence of ASF or F.(LAK/NS=neuraminidase-treated LAK;EXAMPLE-LAK/CAK/NS/ASF/K5=neuraminidase-treated LAK, adherence on CAKIpretreated with ASF, assayed on K562).

FIG. 9 shows additional results of testing effects of asialofetuin (ASF)and fetuin (F) on adherence of NK/LAK cells to HEP2G and CAKI-2 cells at23° C., as in FIG. 7. LAK adherence to HEPG2 (ASGPR+) and CAK.I-2(ASGPR−) in the presence of either ASF or F assayed on RAJI. (LAK=3 dayLAK; R=RAJI targets; FET=fetuin; ASF=asialofetujn; LAK/CAKI/ASF/R=LAK,adherence on CAKI pretreated with ASF, assayed on RAJI).

FIG. 10 shows additional results of testing effects of asialofetuin(ASF) and fetuin (F) on adherence of NK/LAK cells to HEP2G and CAKI-2cells at 23° C., as in FIG. 7. Adherence of neuraminidase-treated LAK toHEPG2 (ASGPR+) and CAKI-2(ASGPR−) in the presence of ASF orF.(LAK/NS=neuraminidase-treated LAK;EXAMPLE-LAK/CAKI/NS/ASF/R=neuraminidase-treated LAK, adherence on CAKIpretreated with ASF, assayed on RAJI).

FIG. 11 shows results of testing effects of cell surface modificationson adherence of NK/LAK cells to HEP2G cell monolayers. Cytotoxicactivity of 5-day LAK, Neuramindase-treated LAK, and Control (no IL-2),assayed on K562.

FIG. 12 shows additional results of testing effects of cell surfacemodifications on adherence of NK/LAK cells to HEP2G cell monolayers, asin FIG. 11. Cytotoxic activity of 5-day LAK, Neuramindase-treated LAK,and Control (no IL-2), assayed on RAJI cells.

FIG. 13 shows additional results of testing effects of cell surfacemodifications on adherence of NK/LAK cells to HEP2G cell monolayers, asin FIG. 11. Adherence of LAK activity to HEPG2 (ASGPR+) after cellsurface modification with neuraminidase, 2,3- or 2,6-sialyltransferases.(EXAMPLE: LAK/HEP/NASE/K5=neuraminidase-treated LAK adhered to HEPG2assayed on K562).

FIG. 14 shows additional results of testing effects of cell surfacemodifications on adherence of NK/LAK cells to HEP2G and CAKI-2 cellmonolayers, as in FIG. 11. Adherence of LAK activity to CAKI-2 (ASGPR−)after cell surface modification with neuraminidase, 2,3- or2,6-sialyltransferases. (Dotted lines in both FIGS. 13 and 14 are thesame controls.)

FIG. 15 shows additional results of testing effects of cell surfacemodifications on adherence of NK/LAK cells to cell HEP2G and CAKI-2monolayers, as in FIG. 11. Adherence of LAK activity to HEPG2 (ASGPR+)after cell surface modification with neuraminidase, 2,3- or2,6-sialyltransferases, assayed on RAJI cells.

FIG. 16 shows additional results of testing effects of cell surfacemodifications on adherence of NK/LAK cells to cell HEP2G and CAKI-2monolayers, as in FIG. 11. Adherence of LAK activity to CAKI-2 (ASGPR−)after cell surface modification with neuraminidase, 2,3- or2,6-sialyltransferases, assayed on RAJI cells.

DETAILED DESCRIPTION OF THE INVENTION

A. Introduction

The present invention is directed to methods for delivering a cell to atarget tissue in a mammal. The methods comprise the steps ofadministering, either simultaneously or sequentially, a carbohydratepresenting molecule (e.g., glycoconjugate) and a cell to the mammal. Inthe methods of the present invention, glycoconjugates, especiallyasialoglycoconjugates, including asialo plasma proteins such asasialoorosomucoid (asialo alpha-(1)-acid glycoprotein), are thought totransiently bind the hepatic ASGP receptor and thereby competitivelyinhibit attachment of cells bearing asialodeterminants from thesereceptors. Without wishing to be bound by theory, hyposialylated anddesialylated proteins/glycoconjugates (also calledasialoglycoconjugates) and cells which bear similar determinants arebound or “trapped” in the liver as a consequence of binding to thehepatic ASGP receptors (see, FIG. 1). Occupation of the receptor by theasialoglycoconjugate inhibits sequestration of the cells bearing similardeterminants of interest in the liver.

In addition, the present disclosure shows that glycoconjugates of theinvention prevent infused cells from concentrating in the alveolarvasculature. This finding suggests that lung sequestration of the cellsmay be related to expression of inflammatory receptors on endothelialcells, analogous to the reperfusion syndrome (see, e.g. Kilgore et al.Cardiovasc Res 28:437-444 (1994) and Eror et al., Clin Immunol90:266-275 (1999). This is supported by reports that orosomucoid, ASOand agalacto/asialo-orosomucoid inhibit neutrophil activation superoxideanion generation, as well as platelet activation as noted above.

The present invention further demonstrates that the glycoproteins may beused to traffic or target cells to particular organs of the body byaltering the particular glycoconjugate administered. The present methodsare useful to improve the efficacy of bone marrow and stem celltransplants, tissue repair, gene therapy or adoptive immunotherapies.

In embodiments wherein the cell is targeted to the lungs, the methodsfeature administering the cell to the mammal in a saline or serumalbumin-saline solution. In some embodiments wherein the hematopoieticstem cell is targeted to the heart, the methods feature administering anasialoorosomucoid, and administering the cell to the mammal. In otherembodiments wherein the mesenchymal stem cell is targeted to the heart,the methods feature administering an orosomucoid, and administering thecell to the mammal. In embodiments wherein the hematopoietic stem cellis targeted to the liver, the methods feature administering anorosomucoid and administering the cell to the mammal. In otherembodiments wherein the mesenchymal stem cell is targeted to the liver,the methods feature administering an asialoorosomucoid and administeringthe cell to the to the mammal. In some embodiments, the orosomucoid orasialoorosomucoid is administered in at least two infusions prior toadministering the cell to the mammal. The methods according to thepresent invention are also useful for inhibiting sequestration of a cellin the liver of a mammal even in the absence of targeting the cell to atarget organ.

Asialoglycoconjugates, for example, asialofetuin and other asialo plasmaproteins, are able to bind to the hepatic parenchyma and Kupffer cellASGP receptors. Blocking these receptors from binding and trapping cellsbearing asialodeterminants, such as bone marrow cells, facilitates andincreases the interval of their systemic circulation. In the case ofbone marrow stem cells, the administration of these compounds preventsthe loss and destruction of bone marrow stem cells and increases theefficiency of engraftment. Bone marrow cells have cell surfaceasialodeterminants capable of binding to the ASGP receptor, and thisbinding can be inhibited by the application of ASGPs.

The present invention takes advantage of the observation that when humanperipheral hematopoietic stem (CD34+) cells or mesenchymal stem cellsare infused into the jugular vein of immunodeficient mice, they localizepredominantly in the lungs. When the cells are preceded by an infusionof asialoorosomucoid, the hematopoietic stem cells predominantlylocalize in the heart, whereas the mesenchymal stem cells localize inthe liver. Alternately, when the cells are preceded by an infusion oforosomucoid (O), the hematopoietic stem cells localize in the liver,whereas the mesenchymal stem cells predominantly localize in the heart.

These protein infusions cause a more quantitative localization into thespecific organs than occurs without them. Furthermore, hematopoieticstem cells that localize in the heart due to the influence ofasialoorosomucoid leave the vascular space and are observed among thecardiac muscle cells by one hour after infusion. Moreover, once in thetissue, these cells lose their CD34 antigen, indicating that they are inthe process of differentiating into cardiomyocytes or heart components(e.g., blood vessels). Additionally, at one hour CD34+ cells have beendemonstrated to move from the vasculature into lung tissue. In anorosomucoid-treated mouse, clusters of stem cells are found in the liverparenchyma and are also demonstrated to lose their CD34 antigen, againsuggesting differentiation into hepatocytes/hepatic or liver parenchyma.

The present invention demonstrates the ability to direct highconcentrations of stem cells to a specific organ in an atraumaticmanner. This enhances the probability and the rate at which stem cellsmigrate into a target tissue and differentiate into the desired celltype. The present invention utilizes the observation that delivery oforosomucoid or ASO to the vessel proximal to the heart causes transfusedstem cells to accumulate in the heart. Without wishing to be bound bytheory, the effect may be caused by the glycoprotein infusionsensitizing the endothelium directly downstream from the infusion site,which causes the endothelial cells to bind stem cells and enhance theirmigration across the endothelium into the tissue.

The present findings with glycoconjugates indicate that the majority ofa stem cell transfusion can be concentrated in the target organ, therebyproviding the means to deliver an effective regimen of cell doses. Thisoffers an opportunity to non-invasively target solid organs such as theheart, thereby competing with invasive direct injection. Perhaps moreimportantly, glycoconjugates provide the means to target very diffusetissues, such as the liver and the kidney, which are not amenable todosage by injection.

It is recognized that hematopoietic stem cells (HSC) recovered from themarrow, peripheral blood or umbilical cord blood and mesenchymal stemcells (MSC) recovered as marrow stromal cells, stromal cells fromliposuction fat, or proliferated from stationary stromal progenitorcells in cord blood-depleted expelled placentas appear to be almostinterchangeable in their differentiation ability, and act as multipotentstem cells.

Such cells have been shown to differentiate into functional cells whenlocalized in specific organs and tissues: hepatocytes and cholangiocytesin the liver, cardiac muscle cells and arterial smooth muscle cells andendothelial cells in the heart, pneumocytes I & II in alveoli andbronchial epithelium in the lungs, chondrocytes for cartilagerestoration, and intestinal mucosal cells, small, medium and large bloodvessels in the heart, etc.

B. Stem Cells

Stem cells may hold the key to replacing cells lost in many devastatingdiseases such as Parkinson's disease, diabetes, acute and chronic heartdisease, end-stage kidney disease, liver failure, and cancer. For manydiseases, there are no effective treatments but the goal is to find away to replace what natural processes have taken away.

To date, published scientific papers indicate that adult stem cells havebeen identified in brain, bone marrow, peripheral blood, blood vessels,skeletal muscle, epithelia of the skin and digestive system, cornea,dental pulp of the tooth, retina, liver, and pancreas. Thus, adult stemcells have been found in tissues that develop from all three embryonicgerm layers.

By way of definition, the following terms are understood in the art:

A “stem cell” is a cell from the embryo, fetus, or adult that has, undercertain conditions, the ability to reproduce itself for long periods or,in the case of adult stem cells, throughout the life of the organism. Italso can give rise to specialized cells that make up the tissues andorgans of the body.

A “pluripotent stem cell” has the ability to give rise to types of cellsthat develop from the three germ layers (mesoderm, endoderm, andectoderm) from which all the cells of the body arise. The only knownsources of human pluripotent stem cells are those isolated and culturedfrom early human embryos and from fetal tissue that was destined to bepart of the gonads.

An “embryonic stem cell” is derived from a group of cells called theinner cell mass, which is part of the early (4- to 5-day) embryo calledthe blastocyst. Once removed from the blastocyst the cells of the innercell mass can be cultured into embryonic stem cells. These embryonicstem cells are not themselves embryos.

An “adult stem cell” is an undifferentiated (unspecialized) cell thatoccurs in a differentiated (specialized) tissue, renews itself, andbecomes specialized to yield all of the specialized cell types of thetissue in which it is placed when transferred to the appropriate tissue.Adult stem cells are capable of making identical copies of themselvesfor the lifetime of the organism. This property is referred to as“self-renewal.” Adult stem cells usually divide to generate progenitoror precursor cells, which then differentiate or develop into “mature”cell types that have characteristic shapes and specialized functions,e.g., muscle cell contraction or nerve cell signaling. Sources of adultstem cells include bone marrow, blood, the cornea and the retina of theeye, brain, skeletal muscle, dental pulp, liver, skin, the lining of thegastrointestinal tract and pancreas.

Stem cells from the bone marrow are the most-studied type of adult stemcells. Currently, they are used clinically to restore various blood andimmune components to the bone marrow via transplantation. There arecurrently identified two major types of stem cells found in bone marrow:hematopoietic stem cells (HSC, or CD34+ cells) which are typicallyconsidered to form blood and immune cells, and stromal (mesenchymal)stem cells (MSC) that are typically considered to form bone, cartilage,muscle and fat. However, both types of marrow-derived stem cellsrecently have demonstrated extensive plasticity and multipotency intheir ability to form the same tissues.

The marrow, located in the medullary cavity of bones, is the sole siteof hematopoiesis in adult humans. It produces about six billion cellsper kilogram of body weight per day. Hematopoietically active (red)marrow regresses after birth until late adolescence after which time itis focused in the lower skull vertebrae, shoulder and pelvic girdles,ribs, and sternum. Fat cells replace hematopoietic cells in the bones ofthe hands, feet, legs and arms (yellow marrow). Fat comes to occupyabout fifty percent of the space of red marrow in the adult and furtherfatty metamorphosis continues slowly with aging. In very oldindividuals, a gelatinous transformation of fat to a mucoid material mayoccur (white marrow). Yellow marrow can revert to hematopoieticallyactive marrow if prolonged demand is present such as with hemolyticanemia. Thus hematopoiesis can be expanded by increasing the volume ofred marrow and decreasing the development (transit) time from progenitorto mature cell.

The marrow stromal consists principally of a network of sinuses thatoriginate at the endosteum from cortical capillaries and terminate incollecting vessels that enter the systemic venous circulation. Thetrilaminar sinus wall is composed of endothelial cells; anunderdeveloped, thin basement membrane, and adventitial reticular cellsthat are fibroblasts capable of transforming into adipocytes. Theendothelium and reticular cells are sources of hematopoietic cytokines.Hematopoiesis takes place in the intersinus spaces and is controlled bya complex array of stimulatory and inhibitory cytokines, cell-to-cellcontacts and the effects of extracellular matrix components on proximatecells. In this unique environment, lymphohematopoietic stem cellsdifferentiate into all of the blood cell types. Mature cells areproduced and released to maintain steady state blood cell levels. Thesystem may meet increased demands for additional cells as a result ofblood loss, hemolysis, inflammation, immune cytopenias, and othercauses. The engraftment efficiency of bone marrow stem cells could beimproved by preventing entrapment by the liver via the hepatic ASGPreceptor.

A “progenitor or precursor” cell occurs in fetal or adult tissues and ispartially specialized; it divides and gives rise to differentiatedcells. Researchers often distinguish precursor/progenitor cells fromadult stem cells in that when a stem cell divides, one of the two newcells is often a stem cell capable of replicating itself again. Incontrast when a progenitor/precursor cell divides, it can form moreprogenitor/precursor cells or it can form two specialized cells.Progenitor/precursor cells can replace cells that are damaged or dead,thus maintaining the integrity and functions of a tissue such as liveror brain.

Means for isolating and culturing stem cells useful in the presentinvention are well known. Umbilical cord blood is an abundant source ofhematopoietic stem cells. The stem cells obtained from umbilical cordblood and those obtained from bone marrow or peripheral blood appear tobe very similar for transplantation use. Placenta is an excellentreadily available source for mesenchymal stem cells. Moreover,mesenchymal stem cells have been shown to be derivable from adiposetissue and bone marrow stromal cells and speculated to be present inother tissues. While there are dramatic qualitative and quantitativedifferences in the organs from which adult stem cells can be derived,the initial differences between the cells may be relatively superficialand balanced by the similar range of plasticity they exhibit. Forinstance, adult stem cells both hematopoietic and mesenchymal, under theappropriate conditions can become cardiac muscle cells. Delineation offull range of potential for adult stem cells has just begun. Stem cellsmay be isolated for transduction and differentiation using knownmethods. For example, in mice, bone marrow cells are isolated bysacrificing the mouse and cutting the leg bones with a pair of scissors.Stem cells may also be isolated from bone marrow cells by palming thebone marrow cells with antibodies which bind unwanted cells, such asCD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), andlad (differentiated antigen presenting cells). For an example of thisprotocol see, Izaba et al., I. Exp. Med. 176-1693 1702 (1992).

In humans, CD34+ hematopoietic stem cells can be obtained from a varietyof sources including cord blood, bone marrow, and mobilized peripheralblood. Purification of CD34+ cells can be accomplished by antibodyaffinity procedures. An affinity column isolation procedure forisolating CD34+ cells is described by Ho et al., Stem Cells 13 (suppl.31: 100-105 (1995). See also, Brenner, Journal of Hematotherapy 2: 7-17(1993). Methods for isolating, purifying and culturally expandingmesenchymal stem cells are known. Specific antigens for MSC are alsoknown (see, U.S. Pat. Nos. 5,486,359 and 5,837,539).

C. Carbohydrate Presenting Molecule

The carbohydrate presenting molecules useful in the present inventioncan be any molecule capable of presenting the appropriate carbohydratestructure that leads to enhancing or inhibiting the targeting of thecell of interest to a target tissue. The targeting function can becarried out using a carbohydrate molecule such as an oligosaccharide,polysaccharide, or the carbohydrate structure can be bound to largermolecule or carrier, referred to here as a glycoconjugate. Typically,the carbohydrate molecule will be linked to either a naturally occurringcarrier (e.g., as part of a glycoprotein or glycolipid) or the carriermay be synthetic (e.g., an engineered polypeptide sequence). One ofskill will recognize that a number of carriers can be used to presentthe appropriate structure. Examples of appropriate carrier moleculesinclude polypeptides, lipids, and the like. Preparation and use oftargeted compounds using asialo carbohydrate moieties is described inthe art (see, e.g., U.S. Pat. Nos. 5,679,323, 5,089,604, 5,032,678 and5,284,646). One of skill will recognize that such compounds can also beused as carbohydrate presenting molecules useful in the presentinvention.

In cases in which the glycoconjugate is a glycoprotein it may begenerally represented by the general formula P-(S)x-Gal wherein P is apeptide residue of a human serum glycoprotein and S is a sugar residueof a human serum glycoprotein; x is an integer from I to 100 and Gal isa galactose residue. Especially useful glycoconjugates include fetuinsand asialofetuins (see, FIG. 2), orosomucoids and asialoorosomucoids andgalactose-bonded polylysine, galactose-bonded polyglucosamine, and thelike.

The methods of the present invention allow cells such as stem cells tobe targeted to such target tissues as the heart, the liver, the kidneysand the lungs, among others. Parenteral administration of aglycoconjugate, such as asialoorosomucoid, may be used to block thehepatic ASGP receptor and allow the cells bearing surfaceasialodeterminants (for example, peanut agglutinin (PNA)+ cells) tocontinue to circulate and migrate to the marrow space. Asialoorosomucoidis one of the glycoproteins which has been shown to bind to the hepaticASGP receptor and has been extensively used to characterize thisreceptor.

Different compounds have different binding affinities for the ASGPreceptor, depending upon the carbohydrate presented (see, FIGS. 3 and4). Thus, one of skill can modulate cell targeting by using compoundsthat present different carbohydrate structures.

Intravenous administration of a glycoconjugate, especially an ASGP suchas asialoorosomucoid, may be used to block the hepatic ASGP receptor andallow the cells bearing surface asialodeterminants to continue tocirculate and migrate to the marrow space or to the organ of interest.The glycoconjugates may be administered to the mammal in any time framerelative to the cells, but in some embodiments, the glycoconjugates areadministered prior to administering the cell. The asialoglycoconjugatesand the cell may be administered in any suitable route, but in someembodiments, they are administered intravenously to the mammal, and inother embodiments, they are administered parenterally. In embodimentswherein the cell is targeted to the lungs, the methods featureadministering the cell to the mammal in a saline or serum albumin-salinesolution. In some embodiments wherein the hematopoietic stem cell istargeted to the heart, the methods feature administering anasialoorosomucoid, and administering the cell to the mammal. In otherembodiments wherein the mesenchymal stem cell is targeted to the heart,the methods feature administering an orosomucoid, and administering thecell to the mammal. In embodiments wherein the hematopoietic stem cellis targeted to the liver, the methods feature administering anorosomucoid and administering the cell to the mammal. In otherembodiments wherein the mesenchymal stem cell is targeted to the liver,the methods feature administering an asialoorosomucoid and administeringthe cell to the to the mammal. In some embodiments, the orosomucoid orasialoorosomucoid is administered in at least two infusions, prior toand after administering the cell to the mammal. The methods according tothe present invention are also useful for inhibiting sequestration of acell in the liver of a mammal even in the absence of targeting the cellto a target organ.

The alpha-(1)-acid glycoprotein (orosomucoid or AAG) is a normalconstituent of human plasma (650±215 μg ml-1) which increases inconcentration as much as fivefold in association with acute inflammationand cancer, and thus is recognized as an acute phase protein.Orosomucoid consists of a single polypeptide chain, has a molecularweight of 44,100, and contains approximately 45% carbohydrate including12% sialic acid. It is the most negatively charged of the plasmaproteins. Certain of the biological properties of orosomucoid arerelated to its sialic acid content. Thus, clearance and immunogenicityof orosomucoid are markedly increased on desialylation. The biologicalfunctions of orosomucoid are largely unknown. Orosomucoid has theability to inhibit certain lymphocyte reactivities includingblastogenesis in response to concanavalin A, phytohaemagglutinin andallogeneic cells, and these inhibitory effects are enhanced inassociation with desialylation. It has been reported thatunphysiologically large (5-15 mg/ml) amounts of orosomucoid inhibit theplatelet aggregation induced by ADP and adrenaline, and there isevidence that a sialic acid-deficient species of orosomucoid appearselevated in several chronic disease states.

D. Gene Therapy

The present invention is also directed to using living cells to delivertherapeutic genes into the body. In some embodiments, the therapeuticgene is a transgene. For example, the delivery cells—a type of stemcell, a lymphocyte, or a fibroblast are removed from the body, and atherapeutic transgene is introduced into them via vehicles well known tothose skilled in the art such as those used in direct-gene-transfermethods. While still in the laboratory, the genetically modified cellsare tested and then allowed to grow and multiply and, finally, areinfused back into the patient. Alternatively, allogeneic cells that bearnormal, endogenous genes can reverse a deficiency in a particular targettissue. Use of cells bearing either transgenes or normal, endogenousgenes is referred to herein as gene therapy.

Gene therapy using genetically modified cells offers several uniqueadvantages over direct gene transfer into the body. First the additionof the therapeutic transgene to the delivery cells takes place outsidethe patient, which allows the clinician an important measure of controlbecause they can select and work only with those cells that both containthe transgene and produce the therapeutic agent in sufficient quantity.

Of the stem cell-based gene therapy trials that have had a therapeuticgoal, approximately one-third have focused on cancers (e.g., ovarian,brain, breast myeloma, leukemia, and lymphoma), one-third on humanimmunodeficiency virus disease (HIV-1), and one-third on so-calledsingle-gene diseases (e.g., Gaucher's disease, severe combined immunedeficiency (SCID), Fanconi anemia, Fabry disease, and leukocyteadherence deficiency).

In view of the foregoing, the methods according to the present inventionare useful for targeting a gene of interest (either a transgene or anendogenous gene) to a tissue in a mammal by introducing a cellcomprising the gene of interest and administering a glycoconjugate tothe mammal. Such methods are useful for treating a disease characterizedby a deficiency in a gene product in a mammal by administering a cellcomprising a functional gene encoding the gene product into the mammaland administering a glycoconjugate to the mammal. Stem cells may be usedas a vehicle for delivering genes to specific tissues in the body. Stemcell-based therapies are a major area of investigation in cancerresearch.

The current invention provides localizing of transfused cells such asstem cells to provide a functional gene to a patient suffering from adisease caused by a lack of that gene. In many instances of geneticallybased diseases, a low level production of that gene product willeffectively ameliorate or cure the disease. By providing the gene thatis deficient through transfusion of stem cells from a normal donor intothe patient, the stem cells may be directed to localize in an organ ortissue of choice, causing a microchimerization of that patient in thatorgan or tissue, from which organ or tissue that gene product can bedelivered to the patient. Therefore, the present invention provides theability to direct the localization of the transfused cells such asallogeneic stem cells that have a stable, normal gene. Such transfusedcells then create a stable micro-chimera of the recipient.

Those of skill in the art are aware of the genetic deficienciescausative of a large array of genetically based diseases. Exemplarygenes and diseases that can be treated include CTFR protein in cysticfibrosis and proteins associated with coagulopathy in the liver. Forexample, treatment of Hemophilia A can be accomplished using genetherapy in such embodiment, a transfusion of such cells as umbilicalcord blood hematopoietic stem cells may be administered to deliver anintact normal Factor VIII gene. Alternatively, transformed cells cancomprise a normal, wild-type Factor VIII gene. Such cells carrying afunctional Factor VIII gene may be directed to localize in the liver,preferably by orosomucoid or asialoorosomucoid perfusion prior to theinfusion of the stem cells. The cells transform into hepatocytes andbegin secreting Factor VIII into the blood.

Other embodiments of gene therapy according to the present inventioninclude treating Hemophilia B (Factor IX deficiency), and antithrombinIII, Protein C, and Protein S deficiencies. While these diseases allinvolve the blood coagulation system, gene therapy may include treatingdifferent tissues, such as muscular dystrophy, cystic fibrosis, and thelike.

E. Introducing Transgenes into Stem Cells

Means for introducing transgenes into cells are well known. A variety ofmethods for delivering and expressing a nucleic acid within a mammaliancell are known to those of ordinary skill in the art. Such methodsinclude, for example viral vectors, liposome-based gene delivery (WO93/24640; Mannino Gould-Fogerite, BioTechniques 6(7):682-691 (1988);U.S. Pat. No. 5,279,833; WO 91/06309; Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7414 (1987); and Budker et al., Nature Biotechnology,14(6):760-764 (1996)). Other methods known to the skilled artisaninclude electroporation (U.S. Pat. Nos. 5,545,130, 4,970,154, 5,098,843,and 5,128,257), direct gene transfer, cell fusion, precipitationmethods, particle bombardment, and receptor-mediated uptake (U.S. Pat.Nos. 5,547,932, 5,525,503, 5,547,932, and 5,460,831). See also, U.S.Pat. No. 5,399,346.

Widely used retroviral vectors include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiencyvirus (Sly), human immunodeficiency virus (HIV), and combinationsthereof. See, e.g., Buchscher et al., J. Virol. 66(5):2731-2739 (1992);Johann et al., J. Virol. 66(5):1635-1640 (1992); Sommerfelt et al.,Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989);Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700, andRosenburg & Fauci, in Fundamental Immunology, Third Edition (Paul ed.,1993)).

AAV-based vectors are also used to transduce cells with target nucleicacids, e.g., in the in vitro production of nucleic acids andpolypeptides, and in vivo and ex vivo gene therapy procedures. See, Westet al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641;Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invst.94:1351 (1994) and Samulski (supra) for an overview of AAV vectors.Construction of recombinant AAV vectors are described in a number ofpublications, including Lebkowski, U.S. Pat. No. 5,173,414; Tratschin etal., Mol. Cell. Biol. (1 l):3251-3260 (1985); Tratschin et al., Mol.Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, Proc. Nail. Acad.Sci. USA 81:6466-6470 (1984), and Samulski et al., J. Virol.63:03822-3828 (1989).

Retroviral vectors are typically used for cells useful in the presentinvention. Such vectors may comprise, for example, an HIV-2 packageablenucleic acid packaged in an HIV-2 particle, typically using a packagingcell line. Cell transduction vectors have considerable commercialutility as a method of introducing genes into target cells. Inparticular, gene therapy procedures, in which the cell transductionvectors of the invention are used to transduce target cells with atherapeutic nucleic acid in an in vivo or ex vivo procedure may be used.Gene therapy provides a method for combating chronic diseases caused bya gene deficiency, infectious diseases such as HIV, as well asnon-infectious diseases such as cancer.

Stem cells such as CD34+ stem cells may be used in ex vivo proceduresfor cell transduction and gene therapy. The present invention utilizesthe feature that stem cells differentiate into other cell types invitro, or can be introduced into a mammal (such as the donor of thecells) where they will engraft in the bone marrow unless targeted toanother organ for differentiation. Hence, the present invention extendsto directing stem cells to particular organs to regenerate tissue suchas to the heart to regenerate cardiac muscle cells, to the lung toregenerate alveoli, and to the kidneys to regenerate tissue and todirecting cells such as CD34+ stem cells to an organ to ameliorate agenetic abnormality by providing efficacious amounts of a deficient geneproduct. Methods for differentiating CD34+ cells in vitro intoclinically important immune cell types using cytokines such a GM-CSF,IFN-γ and TNF-α are known (See, Inaba et al., J. Exp. Med. 176,1693-1702(1992), and Szabolcs et al., 154:5851-5861 (1995)). Yu et al., PNAS 92:699-703 (1995) describe a method of transducing CD34+ cells from humanfetal cord blood using retroviral vectors.

F. Pharmaceutical Compositions

In other embodiments, the present invention provides pharmaceuticalcompositions comprising a cell and a glycoconjugate of the invention.Exemplary glycoproteins include orosomucoids and asialoorosomucoids. Inother aspects, the present invention features kits for treating tissuedamage or for delivering a functional gene or gene product to a tissuein a mammal comprising a cell and a glycoprotein. Stem cells generallyhave been presented to the desired organs either by injection into thetissue, by infusion into the local circulation, or by mobilization ofautologous stem cells from the marrow accompanied by prior removal ofstem cell-entrapping organs before mobilization when feasible, i.e.,splenectomy.

Glycoconjugates may be administered prior to, concomitantly with, orafter infusing the stem cells. In some embodiments, an intravenous fluidbag may be used to administer the glycoconjugate in a saline or dextrosesolution with and without protein, or serum-free media, including, butnot restricted to, RPMI 1640 or AIM-V. In such embodiments, theglycoconjugate may be mixed with the cells in the same bag or in a“piggyback”. The glycoconjugate may also be continued afteradministration of the cells to permit longer systemic circulation timesor increased specific organ accumulation. This procedure may be repeatedas often as needed for delivering a therapeutic dose of the cells to thetarget organ. The preparation may be used with little concern fortoxicity given data from animal studies demonstrating no side effects atdoses of 3-7 mg of glycoconjugate per ml of blood volume (up to 12mg/mouse).

Administration of cells transduced ex vivo can be by any of the routesnormally used for introducing a cell or molecule into ultimate contactwith blood or tissue cells. The transduced cells may be administered inany suitable manner, preferably with pharmaceutically acceptablecarriers. Suitable methods of administering such cells in the context ofthe present invention to a patient are available, and, although morethan one route can be used to administer a particular composition, aparticular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.Parenteral administration is one useful method of administration. Theformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and in some embodiments, can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water, forinjections, immediately prior to use. These formulations may beadministered with factors that mobilize the desired class of adult stemcells into the circulation.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.Cells transduced by the vector as described above in the context of exvivo therapy can also be administered parenterally as described above,except that lyophilization is not generally appropriate, since cells aredestroyed by lyophilization.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular cells employed and the condition of thepatient, as well as the body weight of the patient to be treated. Thesize of the dose also will be determined by the existence, nature, andextent of any adverse side effects that accompany the administration ofa cell type in a particular patient. In determining the effective amountof cells to be administered in the treatment or prophyLAKis of diseases,the physician should evaluate circulating plasma levels, and, in thecase of replacement therapy, the production of the gene product ofinterest.

Transduced cells are prepared for reinfusion according to establishedmethods. See, Abrahamsen et al, J. Clin. Apheresis 6-48-53 (1991; Carteret al., J. Clin. Apheresis 4:113-117 (1988); Aebersold et al., J.Immunol. Methods 112:1-7 (1988); Muul et al., J. Immunol. Methods 101:171-1 81 (1987) and Carter et al., Transfusion 27:362-365 (1987). Aftera period of about 2-4 weeks in culture, the cells may number between1×10⁶ and 1×10¹⁰. In this regard, the growth characteristics of cellsvary from patient to patient and from cell type to cell type. About 72hours prior to reinfusion of the transduced cells, an aliquot is takenfor analysis of phenotype, and percentage of cells expressing thetherapeutic agent.

For administration, cells of the present invention can be administeredat a rate determined by the LD-50 of the cell type, and the side effectsof the cell type at various concentrations, as applied to the mass andoverall health of the patient. Administration can be accomplished viasingle or divided doses. Adult stem cells may also be mobilized usingexogenously administered factors that stimulate their production andegress from tissues or spaces, that may include, but are not restrictedto, bone marrow or adipose tissues. The exemplary glycoconjugates may beadministered concurrently, prior to and/or following stem cellsmobilization, or at a time when the amount of cells in the peripheralcirculation is optimal for the desired therapeutic endpoint.

G. Adoptive Immunotherapy

It has already been shown that intravenously administered LAK cells aresequestered predominantly in the lungs and the liver (Lotze, M. T., etal., The in vivo distribution of autologous human and murine lymphoidcells grown in T cell growth factor (TCGF)-Implication for the adoptiveimmunotherapy of tumors. J. Immunol. 125: 1487-1493 (1980) (possibly dueto interaction of asialodeterminants on the LAK cell surface with ASGPreceptors on the surfaces of endothelial cells, Kupffer cells, andhepatocytes (Kolb et al., 1979; supra; Kolb-Bachofen, et al., 1984,supra) and that metastatic tumors in these organs can be dramaticallyreduced by LAK therapy (Rosenberg, 1987, supra). Intravenously injectedmurine bone marrow cells, neuraminidase-treated lymphocytes, naturalkiller (NK), and LAK cells all share this same trafficking pattern(Samlowski et al., 1984, supra; Samlowski et al., 1985, supra; Kolb etal., 1979, supra; Kolb-Bachofen, et al., 1984, supra; Rolstad. B. etal., Natural killer cell activity in the rat V. The circulation patternsand tissue localization of peripheral blood large granular lymphocytes(LOL), J. Immunol. 136: 2800-2808 (1986); Rosenberg, 1987, supra).Moreover, all these cells have asialodeterminants on their surface.Kradin, R. L., et al., Tumor-derived interleukin-2-dependent lymphocytesin adoptive immunotherapy of lung cancer. Cancer Immunol. Immunother 24:76-85.(1987), have gamma-camera imaged patients that have received¹¹¹In-labeled tumor-derived interleukin-2-dependent lymphocytes (derivedfrom metastatic adenocarcinoma of the lung). These T “killer” cellsderived from human tumors also migrate to the liver and lungs. Based onthis preferential localization in the liver of human LAK cells and theirability to kill hepatocellular carcinoma, Hsieh et al., Lysis of primaryhepatic tumors by lymphokine activated killer cells. Gut 28:117-124(1987), have conducted Phase I trials for the treatment of this tumor;It has also been suggested that for treatment of liver tumors thatselective administration of LAK cells with IL-2 via a catheter insertedinto the hepatic artery should be an effective means of administrationwhich may decrease the magnitude and scope of side effects (Fagan. E.A., et al., Immunotherapy for Cancer: the use of lymphokine-activatedkiller (LAK) cells. Gut 28:113-116 (987)).

Human, rat, and mouse liver have been shown to specifically sequester,trap, or “clear” desialylated serum glycoproteins (eg.,asialotransferrin) by recognition of galactose residues made terminal bythe removal of sialic acid (i.e., asialogycoproteins) and ageddesialylated erythrocytes via high affinity hepatic asialoglycoproreinreceptors (Ashwell, G. The role of cell-surface carbohydrates in bindingphenomena. In: Mammalian Cell Membranes, Vol. 4, Butterworth, London, OX(1977); Asbwell, G., et al., Carbohydrate-specific receptors of theliver. Ann. Rev. Riochem. 51: 531-554 (1982); Harford et al., Thehepatic receptor for asialoglycoproteins. In: The Glycoconjugates; Vol.4; Part B (ed. M. I. Horowitz) Academic Press, New York, 1982). Thehuman, rat and rabbit ASGP receptors display virtually identicalcharacteristics: specificity, cation requirements, pH optimum, affinity,subunit size, and temperature dependent internalization of the receptorand degradation of the asialoligand (Dunn et al., Low temperatureselectivity initibits fusion between pinocytotic vesicles and lysosomesduring heterophagy of ¹²⁵I-asialofetuin by the perfused rat liver. J.Biol. Chem. 225 5971-5978 (1980); Schwartz et al., Characterization ofthe ASGP receptor in a continuous hepatoma line. J. Biol. Chem 256:888-8881 (1981); Ashwell et al., 1982, supra; Mueller et al.,Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes:receptor-positive and receptor negative endosomes. J. Cell. Biol.102:932-947 (1986). At 5-20° C. the ligand receptor complex is notinternalized; whereas, at 37° C. this complex was internalized anddegraded and the receptor recycled to the cell surface undamaged Muelleret al., 1986, supra). On average a cell containing 225,000 receptors caninternalize approximately 30,000 soluble ligand molecules per cell perminute; each functional receptor can bind and internalize one ligandevery 8 minutes (Schwartz et al., 1982, supra). Hepatocytes shareasialo- or GalNAc/Gal-specific receptors with Kupffer cells, and liverendothelial cells (Kolb-Bachhofen et al., 1984, supra) and the hepatomaline, HEPG2 has been well characterized with respect to this receptor(Schwartz et al., 1981, supra). There are 150,000 high affinity sitesper HEPG2 cell and 500,000 per normal hepatocytes; the K_(d) of about7×10⁹ M is the same for both. Thus, using adherence to cell line such asHEPG2 with a well-characterized asiaioglycoprotein receptor, as an invitro correlate of in vivo adherence (as in the EXAMPLES, below) is acost effective and simple system in which to determine parameters andpossible problems that will be encountered in the in vivo traffickingstudies.

Parenteral administration of asialoglycoconjugates (e,g., asialofetuin)to block asialogycoprotein receptors has been shown to increase theefficiency of bone marrow engraftment 5- to 10-fold by blocking hepaticsequestration of these cells by blocking hepatic ASGP receptors(Samlowski et al., 1984, supra). Given that LAK cells haveasialodeterminants on their surfaces, as shown by the in vitro studiesherein (see Examples 5-16), then they also most likely are taken up orsequestered in the liver via the ASGP receptors. This would result in anet loss of circulating numbers of LAK effector cells that would beavailable to participate in the reduction or lysis of tumors.Sequestered LAK cells might not reach the tumor. According to thepresent invention, by blocking the hepatic ASGP receptors hepaticsequestration can be prevented. Ultimately, the efficacy of LAK therapywould be improved by eliminating hepatic sequestration of these cells bythe intravenous administration of asiaioglycoconjugates or bymodification of the LAK cell surface with sialidases orsialyltransferases. This would allow fewer LAK cells or fewer cycles ofLAK therapy or even less IL-2 to be used during the therapy, therebyreducing the toxicity associated with LAK therapy.

In theory, LAK therapy should be one of the safest and least toxictherapies in the treatment of cancer; however, it has not metexpectations (Rosenberg, 1987, supra; Durant, Immunotherapy of cancer:The end of the beginning? N. EngI. J. Med. 316:939-940 (1987)). LAKcells have also been shown to kill unmodified normal cells, includingnormal lymphocytes, endothelial cells, and hepatocytes, by someinvestigators, but not by others. The present invention improves theefficacy of LAK therapy by increasing the number of circulating “killer”cells and thereby improving the probability that these cells willencounter tumor cells located in the periphery, instead of beingprimarily sequestered in the liver. Eliminating hepatic sequestrationshould therefore improve the response rate of LAK therapy for tumorslocated in organs other than liver. Preventing hepatic sequestration ofLAK cells should also decrease the severe toxicity and the liver damageassociated with this therapy. In addition, this will also reduce thepossibility of permanent damage caused by the autoimmune destruction ofthe liver parenchyma by trapped lymphocytes (Kolb-Bachofen et al., 1979,supra; Anderson et al., Toxicity of human recombinant interleukin-2 inthe mouse is mediated by interleukin-activated lymphocytes. Lab.Investigation 59:598-612 (1988).

EXAMPLES Procedures

Intravenous cannulas were placed into the external jugular vein ofNOD-SCID mice under anesthesia (Institutional Animal Care and UseCommittee protocol #AM87046-07) to enable the efficient delivery of¹¹¹In-labeled stem cells i.v. Tylenol elixir was administered by mouthafter recovery from anesthesia. Briefly radiolabeled CD34+ cells weretaken up in 100-250 ul of 5% human plasma albumin in saline and injectedinto the cannula and then flushed with 50 ul of the albumin-saline. Themice were imaged by nuclear medicine.

Mice: NOD-SCID, female mice (Nonobese diabetic/LtSz-scid/scid) wereobtained from the Jackson Laboratory, Bar Harbor, Me. at 1-2 months ofage. These animals were maintained in microisolator cages in a specialisolator room. The air was HEPA filtered, and the animals were changedin a laminar flow hood within the facility. All food, bedding, and waterwas sterilized. NOD-SCID mice were ideally suited for the study ofxenotransplanted tumors and hematopoietic cells and lymphocytes becauseof their immunoincompetence including greatly reduced NK activity. See,e.g. Hogan, et al., Biology of Blood & Marrow Transplantation 3:236-246(1997); Noort, et al., Bone Marrow Transplantation 22 Suppl 1:S58-60(1998).

All administrations of agents or cells were done either i.v. or i.p.

Stem Cells: CD34+ stem cells were isolated from apheresis stem cellcollection products derived from deceased cancer patients. They werepurified to 95-99% purity using antibody conjugated to CD34 conjugatedto magnetic beads (MACS separation columns; Miltenyi Biotec, Auburn,Calif. and cryopreserved.

Human mesenchymal stem cells (hMSCs; PT-2501) obtained through a FDAmonitored paid bone marrow donor program were purchased from PoieticsTechnologies, BioWhittaker (Walkerville, Md.). The cells were thawedaccording to manufacturer recommendations, resuspended, and radiolabeledin Mesenchymal Stem Cell Basal Medium (MSCBM).

Proteins administered: Orosomucoid (alpha-1 acid glycoprotein) andasialoorosomucoid (ASO) were administered in the following buffercontaining 0.16 mM Caprylate. 10 mM TRIS, 150 mM NaCl, pH 7.0.

Anesthesia & analgesia: A rodent anesthesia cocktail of 0.04 ml per20-30 g mouse i.p. (Rodent Cocktail recipe: 1.5 ml of 50 mg/ml ketamine,plus 1.5 ml of 20 mg/ml xylazine, plus 0.5 ml of 50 mg/ml acepromazine)was used. The anesthetic agent, Rodent anesthesia cocktail, wasadministered i.p. as follows:

1) for surgery—0.04 ml per 20-30 g mouse, and

2) for imaging—0.02 ml per 20-30 g mouse.

Post-surgical Analgesia: Tylenol 60 ul/20 g mouse (6.10 mg) wasadministered by mouth after anesthesia had partially worn off. Theanalgesic agent was Tylenol by mouth at 60 ul (6.10 mg) per 20 g mouseimmediately after surgery or at the first signs of distress. Xylazinecontained in an anesthetic formulation may also act as an analgesic.

Surgical procedure (Standard cannula placement): After anesthetizing theanimals as previously described, the threads for suturing a cannulafilled with citrate saline were soaked in 70% ethanol. The anesthetizedanimals were secured with paper tape on the operating platform ventralside up. The area from just below the clavicle to the ear was shaved.The shaven area was cleaned with Betadine and rinsed with 70% ethanol. Avertical incision was made in the skin of the right neck from the top ofthe rib cage to the jaw bone to expose the stemocleidomastoid musclewith the external jugular vein just beneath. To clearly expose theoperating field, the skin was retracted with wire hooks (secured tosmall weights). Retraction should not distort the underlying tissue butshould stabilize the area for visualization and cannula insertion. Thevein was cleared of overlying fat and fascia using microscopic forceps.The circulation in the superior vena cava was cut off using a half aknot of 4 O silk surgical sutures. One side of the thread was securedwith a clamped hemostat. A second piece of thread was looped around thebottom of the vein to make a half knot without pulling it tight. Thisloop was used to secure the cannula once it had been inserted into theexternal jugular vein. The surface of the vein was nicked with themicroscissors. The cannula was inserted into the vein with beveled sideup. The cannula was slid down diagonally until the anchor was flush withthe wall of the vein and the lower knot tightened. The cannula wastested by pushing saline through it. The lower knot was finished afterverifying no leakage. A full knot was tied around the cannula using thetop thread. Saline flow in the cannula was monitored. The top thread wasused to go under, catching tissues, and a knot was tied over the cannulaagain with this thread. A full knot was made using an end of the topthread and the bottom thread. This secures the superior and inferiorthreads over the hub of the cannula to prevent accidental dislodgement.The cannula was clamped off and the syringe removed. The cannula waspositioned underneath the skin of the neck and exteriorized just belowthe occiput at the nape of the neck while rotating the animal (dorsalside up). An autoclip was used to staple the heat shrink part of thecannula in place near the exit. The cannula was cut to a reasonablelength (1.5-2.0 inches), and a wire plug was placed into it. The animalwas turned over to its original position and the neck closed withautoclip being careful not to puncture the cannula.

Surgical procedure (Da Vinci Microport Vascular System cannulaplacement): The Da Vinci Microport Vascular System (Da Vinci Biomedical,South Lancaster, Mass.) is a closed injection route permitting itsimplantation up to 2 weeks prior to trafficking experiments without lossof patency. The essential difference is that the port is notexternalized as before. This eliminates additional risk forcontamination and damage to the cannula caused by chewing andscratching.

The incision area was cleaned with Betadine prior to initial cuts. Themouse was then taped (back side up) to the surgery board. An incision3-4 mm was made. Next, the incision was made on the chest 4-5 mm. Atunnel was made from the back incision to the front incision in order tofeed the cannula through the back to the chest. Heparin was pushedthrough the cannula. The cannula was then pulled through using thehemostats. The skin was pulled loose from the tissue on the back forplacement of the port. The port was sutured down to the tissue in themiddle upper neck area. It was sutured in two places using a triple knottie. Next, the mouse was turned on its back with its chest up. Thecannula was then cut at an angle, where at least 1 mm and at most 2 mmof cannula was inserted in the jugular vein. The jugular vein wasisolated in the chest after some fat and tissue was pulled away. Thearms of the mice were taped down on their sides because that pushes thechest forward and further exposes the jugular vein. Once the jugularvein was isolated, two sutures were placed around it. The top of thevein was tied off enough to slow the flow of blood, but not tocompletely stop the flow. The lower tie was one to 2 mm from the top,and it was not tightened. The lower tie was used later to hold thecannula in place and to stop excessive bleeding from the jugular vein.Next, a small cut was made in the jugular vein between the two ties, sothat the cannula could be fed into the vein. Once the cannula was placedin the vein the lower tie was tightened around the cannula within thevein. Next, the cannula was checked for leaks by running heparin throughthe cannula. After verifying no leaks, both incisions were closed.

¹¹¹Indium Oxine Labeling Procedure: ¹¹¹In-oxine labeling of adult humanCD34+ or mesenchymal stem cells (hMSCs) was performed using amodification of the Amersham Healthcare Procedure for labelingautologous leukocytes.

Harvesting for tissues for histopathology: Tissues were harvested aftereuthanasia. After the 1-hour-image, the organs were harvested and halfthe organ was fixed in 10% neutral buffered formalin and the other halfwas frozen in OTC for frozen sections. The images presented herein arefrom fixed tissues.

Necropsy Procedure For Collection of Mouse Tissues: An initial midlineskin incision from the anterior cervical region to the brim of the pubiswas made followed by an abdominal incision following linea alba from thesternum to the pubis with a lateral reflection of the abdominal wall byincision following the caudal ribs. The sternum was reflected anteriorlyby cutting the ribs at approximately the level of the costochrondraljunction, incising the diaphragm and pericardium as needed. Anteriorly,reflection of sternum was extended to include the ventral cervicalmuscles to expose the trachea. The trachea and esophagus were incised atthe mid cervical area and reflected caudally, cutting attachments asnecessary to remove the thoracic viscera in toto. Following removal ofthe thoracic viscera, the entire heart was dissected free and immersedin 10% neutral buffered formalin. After immersion, the heart wasmassaged lightly with serrated tissue forceps to force fixative into thecardiac chambers. The trachea with attached lung was then immersed infixative without further dissection. The spleen was visualized, omentalattachments incised, removed and immersed whole in formalin fixative.The stomach and intestinal tract were removed by incising the rectum andreflecting the viscera anteriorly while cutting attachments asnecessary. The liver was removed in toto and immersed whole in formalinfixative. The kidneys were removed and immersed whole in formalinfixative. The pancreas was incised from the anterior duodenum andimmersed in formalin fixative.

Trimming of Tissues for Paraffin Processing and Microtomy: The heart wasplaced on the trimming board with the right ventricle on the uppersideand the left ventricle on the underside next to the trimming surface. Asingle upper to lower incision was made through the right ventricle andatrium and great vessels at the base of the heart continuing through theinterventricular septum and the left cardiac chambers to achieve twoapproximately equal halves. Each half was placed into separate embeddingcassettes containing fixative saturated foam pads and labeled “heart1”and “heart2”. The entire left and right lungs were separated frommidline tissues and placed flat on fixative-saturated foam pads incassettes labeled left and right lung. Liver sections were taken fromthe right lateral and medial liver lobes and placed into anappropriately labeled cassette. The left lateral and medial lobes weresectioned and handled in a similar manner. The entire spleen was placedin an appropriately labeled embedding cassette and oriented with onelong margin down, taking advantage of the curvature to increase initialsectional area. For one kidney, a whole coronal section was taken fromthe midpoint of the kidney. The remaining kidney was sectionedlongitudinally. Both sections were placed in a single cassette. Thecollected pancreas was placed on formalin-saturated foam pad in anappropriately labeled cassette.

Imaging procedures: Nuclear Medicine. NOD-SCID mice were anesthetizedusing rodent anesthesia cocktail. Once anesthetized, the mice wereplaced on a foam hemi-cylindrical mouse positioning device (MPD) andcovered with a tube sock. The MPD allows better visual separation of thelungs and liver as compared to placing the mouse on a flat surface. Thefoam on which the mouse was placed, and the tube sock coveringmaintained a comfortable temperature permitting longer imaging withoutadditional anesthesia. The MPD was placed on a narrow table between thedual heads of a Siemens E.Cam Gamma Camera and imaged statically ordynamically in 2-D or SPECT. ⁵⁷Co-Spot Marker is used to mark anatomicpositions (nose, tail, cannula, etc.). The data was analyzed using aSiemens ICON system for regions of interest or percent of injected dose(e.g. liver, spleen, heart).

CT imaging: A CT scan was performed (G. E. Medical System High SpeedSpiral Tunnel) for tumor assessment and to enable theregistration/alignment of the nuclear medicine image with that of the CTin order to determine precise location of injected radiolabeled stemcells using the method described by Arata L., Clinical Uses for MedicalImage Registration: Experiences at Three Hospitals. Proceedings ofPACMEDTec Symposium in Honolulu, Hi., Aug. 17-21, 1998 and Nelson, etal., Electromedica 68 (2000) 45-48. CT scans were performed during anuclear medicine imaging session while the animals were underanesthesia. Anesthetized animals were transported to CT, either justprior to or immediately after, the nuclear medicine scan. Usually onlyone CT was done per animal. CT was used to precisely localize theradiolabeled materials anatomically, by fusing the CT image with that ofthe nuclear medicine SPECT images.

Gamma camera imaging using a Siemens E. Cam dual head gamma cameramonitored the in vivo trafficking patterns of all human stem cellsdescribed in the following examples. Mice were placed on a MousePositioning Device (MPD) and placed between the detectors on the imagingplatform.

Example 1

ASO Administered I.V. Directs Human CD34+ to the Heart

Asialoorosomucoid (ASO)/High Dose HSC: When an infusion of 5.75×10⁶ HSCwas preceded by 3.3 mg ASO, 77±1% of the infused cells were found in theheart immediately after infusion, 75±5% remained in the heart region at1.5 hr, decreasing to 52±1% at 24 hr.

5.75×10⁶ ¹¹¹In-labeled human CD34+ (hCD34+) peripheral blood stem cellswere administered intravenously (i.v.) via an external jugular veincannula to 2 month old, NOD-SCID, female mice (Non-obesediabetic/LtSz-scid/scid) obtained from the Jackson Laboratory, BarHarbor, Me. The radiolabeled CD34+ stem cells were administered afterpretreatment of the mouse with 3.3 mg of asialoorosomucoid (ASO) i.v.The in vivo trafficking patterns were followed by gamma camera imagingusing a Siemens E.Cam dual head gamma camera from immediately afterinjection up to 36 hr postinfusion. Human CD34+ were isolated fromapheresis stem cell collection products derived from deceased cancerpatients. They were purified to 95-99% purity using antibody conjugatedto CD34 conjugated to magnetics beads (MACS) separation columns;Miltenyi Biotec, Auburn, Calif. and cryopreserved.

Radiolabeled CD34+ stem cells administered after ASO migratedimmediately to the heart. Anatomic localization was facilitated by theuse of a ⁵⁷Co-point source positioned at the level of the cannula. Up to79.2% of the injected dose was located in the heart at 1.5 hours. Thesecells did not migrate to the liver and spleen early in the postinfusionfollow up images but could be found in the liver later after 24 hours.However, 51.6-53.2% of the originally injected dose remained in theheart at 24 hours. At 36 hours imaging was conducted with the cannula invivo and with the cannula removed and placed next to the sacrificedanimal. These images show that the injected cells were not trapped inthe cannula but were actually in the heart.

Example 2 O Administered I.V. Enables Human CD34+ Cells to Migrate tothe Liver and Spleen but not to the Heart

Orosomucoid/High Dose HSC: When an infusion of 5.75×10⁶HSC was precededby 5.5 mg orosomucoid, 74±3% of infused cells were found in the liverand spleen immediately after infusion, 74±4% of the cells remained inthe liver region at 1.5 hr, decreasing to 63±1% at 24 hr.

The preparation and procedures set forth in Example 1 were repeated.

5.75×10⁶ ¹¹¹In-labeled human CD34+ (hCD34+) peripheral blood stem cellswere administered intravenously (i.v.) via an external jugular veincannula to 2 month old, NOD-SCID, female mice (Non-obesediabetic/LtSz-scid/scid) obtained from the Jackson Laboratory, BarHarbor, Me. The radiolabeled CD34+ stem cells were administered afterpretreatment of the mouse with 5.5 mg of orosomucoid (O) i.v.

Mice were imaged and the biodistribution of the radiolabeled hCD34+cells monitored as described in Example I. Radiolabeled hCD34+administered after O migrated immediately to the liver/spleen area andremained there until 36 hours. Anatomic localization was facilitated bythe use of a ⁵⁷Co-point source positioned at the level of the cannula.The localization to the liver/spleen region ranged from 76.3%immediately postinfusion to 63.6% at 24 hours. No ¹¹¹In-labeled cellswere found in the region of the heart.

At 36 hours imaging was conducted with the cannula in vivo and with thecannula removed and placed next to the sacrificed animal. These imagesshow that the injected cells were not trapped in the cannula.Radioactivity was found at or below the cannula placement, i.e., in theregion of the liver/spleen.

Example 3 O Enables Hcd34+ Cells to Migrate to the Liver/Spleen WithoutSignificant Migration to the Heart

Orosomucoid/Low Dose HSC: When an infusion of 0.5×10⁶ HSC (one-tenth theprevious cell dose) was preceded by 11 mg orosomucoid, 43±2% of infusedcells were found in the liver and spleen immediately after infusion, and40±3% of the cells remained in the liver region at 1 hr.

The preparation and procedures set forth in Example I were repeated.0.5×10⁶ ¹¹¹In-labeled human CD34+ (hCD34+) peripheral blood stem cellswere administered intravenously (i.v.) via an external jugular veincannula to 2 month old, NOD-SCID, female mice (Non-obesediabetic/LtSz-scid/scid) obtained from the Jackson Laboratory, BarHarbor, Me. The radiolabeled CD34+ stem cells were administered afterpretreatment of the mouse with 11.0 mg of orosomucoid (O) i.v.

Mice were imaged and the biodistribution of the radiolabeled hCD34+cells monitored as described above. Approximately 1 hour after infusion,the mice were sacrificed and the organs were harvested, and half of theorgan was fixed in 10% neutral buffered formalin. Tissue sections wereexamined microscopically after immunohistochemical staining for humanCD34 and in situ hybridization for the visualization of human DNA.Nuclear medicine monitoring for the first ten minutes and 1 hourpostinfusion showed that the radiolabeled hCD34+ cells localized to theregion of the liver/spleen.

Microscopic examination of the heart after immunohistologic staining forCD34 demonstrated hCD34+ cells in the endocardial blood vessel. A fewhCD34+ cells could be seen in the lung in the alveolar septum. Clustersof cells with stem cell morphology could be seen in the hepaticsinusoid. In situ hybridization for human DNA clearly showed that hCD34+cells were not found in the heart muscle or interventricular septum butwere present in the lung.

Example 4 AS0 Followed by O Directs hCD34+ Cells to the Heart and Lungbut not the Region of the Liver/Spleen

Asialcorosornucoid (ASO)+Orosomucoid/Low Dose HSC. When infused ASOcaused HSC to localize in the heart, the protocol was changed to havethe ASO bolus chased with a bolus of orosomucoid, to test whether theaccumulation in the heart would be maintained. HSC were againconcentrated in the heart when an infusion of 0.5×10⁶ HSC was precededby 3.3 mg ASO, then 5.5 mg orosomucoid. This caused 44±5% of the infusedcells to accumulate in the heart immediately after infusion. 37±3% ofthe infused cells remained in the heart region at 1 hr. The localizationin the heart was the major concentrated signal from the cells, althoughthe percent of infused was reduced from the ca. 75% seen in Example 1.

The preparation and procedures set forth in Example I were repeated.0.5×10⁶ ¹¹¹In-labeled human CD34+ (hCD34+) peripheral blood stem cellswere administered intravenously (i.v.) via an external jugular veincannula to 2 month old, NOD-SCID, female mice (Non-obesediabetic/LtSz-scid/scid) obtained from the Jackson Laboratory, BarHarbor, Me. The radiolabeled CD34+ stem cells were administered afterpretreatment of the mouse with 3.3 mg of ASO i.v. followed by 5.5 mg 0i.v.

Mice were imaged and the biodistribution of the radiolabeled hCD34+cells monitored as described in Example 1. Nuclear medicine monitoringfor the first ten minutes and 1 h postinfusion showed that theradiolabeled hCD34+ cells localized to the heart.

Approximately 1 hour after infusion, the mouse was sacrificed and theorgans were harvested and half the organ was fixed in 10% neutralbuffered formalin.

Microscopic examination of the heart after immunohistologic staining forCD34 revealed clusters of hCD34+ cells in the interventricular septum,and cells within those clusters that were morphologically similar to thestained cells but that were CD34 negative. These images reflected thebiodistribution depicted by nuclear medicine studies. The presence ofhCD34+ cells in the heart was dramatically demonstrated by in situhybridization. Both immunohistochemical staining for CD34 and in situhybridization for human DNA demonstrated that the infused stem cellslocalized to the lung and could be readily seen in the alveolar septa,blood vessels, and other structures. Detection of human DNA revealed thepresence of many more cells in the lung and heart than would have beenpredicted by CD34 staining. No hCD34+ cells or cells morphologicallyresembling hCD34+ cells were found in liver, spleen or kidney.

Example 5 HSC Administered in 5% Human Serum Albumin (WithoutOrosomucoid or ASO) Migrated Predominantly to the Lungs

Plasma Albumin/High Dose HSC: When HSC were administered through thecatheter without prior protein infusion, 78±3% of infused cells werefound in the lungs at 0 hr, 54±10% at 1 hr, and 50±13% at 12 hr.Histological examination of lungs of mice similarly treated,demonstrated infused cells within the alveolar septa and thevasculature.

2.7×10⁶ ¹¹¹In-labeled HSC were administered intravenously (i.v.) via acannula implanted in the external jugular vein of a two-month old,female NOD-SCID mouse in 0.1 ml saline containing 5% human serumalbumin. Mice were imaged and the biodistribution of the radiolabeledhCD34⁺ cells monitored as described in Example 1.

Radiolabeled HSC, administered in saline containing 5% human serumalbumin, migrated immediately to the lungs. Anatomic localization of thelabeled cells was facilitated by the use of a ⁵⁷Co-point sourcepositioned at the level of the cannula exit site below the scapulae andnose. Moreover, the position marker at the cannula was verified to be atthe diaphragm by CT whole body scans, transverse and coronal sections.The clip at the cannula exit site served as a landmark. The lungs werevisualized below the nose marker and above the cannula marker arid theliver and spleen below the cannula marker. Up to 95.4% of the injecteddose was located in the lungs at initial imaging (Table 1). In four micethe values for the lungs ranged from 52.6-95.4% of whole bodyincorporation for the initial imaging time points. At 1 h, HSC werelocated predominantly in the lungs with some counts visible in the bloodcirculation. In one mouse at 1h some localization was seen below thecannula marker, which may have been liver and spleen; however, theoutline was indistinct. At 12 hr in that mouse, radiolabeled CD34⁺ stemcells were found in the liver/spleen region. However, more than 34.7%(range 34.7-68.5%) of the originally injected dose remained in the lungsof other animals imaged at 12 h.

While the localization to the lungs immediately after injection (initialor Oh time points) varied from animal to animal, the percent of theoriginal localization to the lungs remaining at subsequent scans wasmore constant. Using the dorsal images at 1h, 72.1-75.5% of the cellsinitially localized in the lung were retained in the lung region. Usingthe dorsal images at 12 h, 78%, 72.1% and 50.5% of the initial lungincorporation remained in the lungs of the three mice imaged.

Example 6 Orosomucoid Directs MSC to the Heart

Orosomucoid/Low Dose MSC: When a human mesenchymal stem cell infusion(0.56×10⁶ cells) was preceded by 11 mg orosomucoid, 68±7% of infusedcells were found in the heart at 0 hr, and 61±3% at 1 hr.

MSC were obtained from BioWhittaker, (Poietics Division, cryopreservedPT-2501 >750,000 cells per ampoule) and labeled with ¹¹¹In as inprevious examples, except that the MSC were labeled, washed, andinjected in Basal Stem Cell Medium (Poietics) containing 5% human serumalbumin (HSA). 0.56×10⁶ ¹¹¹In labeled, human mesenchymal stem cells(MSC) were administered via an implanted Da Vinci Microport VascularSystem cannula in the external jugular vein of a two-month old, femaleNOD-SCID mouse in 0.21 ml of basal stem cell medium containing 5% humanserum albumin (HSA). Immediately prior to administration of MSC, 11.0 mgof orosomucoid was administered i.v. in 0.2 ml.

Mice were imaged and the biodistribution of the radiolabeled MSCs cellsmonitored as described in Example 1. Gamma camera monitoring initially(0 hr) and at 1 hr post-infusion showed that the radiolabeled MSClocalized to the region of the heart. Region of interest analysis of theimages revealed that approximately 61.7-75.5% of the injectedradioactivity initially localized to the heart and at 1 hr approximately58-64% of the infused cells remained in this region. The positions ofthe cannula, diaphragm, heart, lungs, and liver were verified by CTscans (coronal sections). In situ hybridization showed human cellspredominantly in the heart, but not the liver.

Example 7 ASO Followed by Orosomucoid Directs MSC to the Liver/Spleen

MSC were obtained from BioWhittaker, (Poietics Division, cryopreservedPT-2501 >750,000 cells per ampoule) and labeled with ¹¹¹In. As inExample 6, the MSC were labeled, washed, and injected in Basal Stem CellMedium (Poietics) containing 5% human serum albumin (HSA).

Asialoorosomucoid (ASO)+Orosomucoid/Low Dose MSC: This example wasdesigned to compare the trafficking of MSC with HSC (Example 4) at thelow cell dose, so the sequential infusion of ASO and orosomucoid used inExample 4 was applied. A human mesenchymal stem cell infusion (0.56×106cells) was preceded by 4.3 mg ASO followed by 5.5 mg orosomucoid. 63±5%of the infused cells were found in the liver and spleen at 0 hr, and57±7% at 1 hr.

0.56×10⁶ “In-labeled, MSC were administered i.v. in 0.21 ml of basalstem cell medium containing 5% human serum albumin (HSA). Prior toadministration of MSC, 0.1 ml containing 4.3 mg of ASO, followed by 0.1ml containing 5.5 mg orosomucoid were administered i.v. The ASO,orosomucoid and MSC were administered via an implanted Da VinciMicroport Vascular system cannula in the external jugular vein of atwo-month old, female NOD-SCID mouse.

Mice were imaged and the biodistribution of the radiolabeled MSCmonitored as in Example 1. Gamma camera monitoring initially and at 1 hpost-infusion showed that the radiolabeled MSC localized to the regionof the liver/spleen. Region of interest analysis of the initial imagesrevealed that approximately 59.2-66.7% of the injected radioactivitylocalized to the liver/spleen and at 1 h approximately 51.9-61.1% of theinfused cells remained in this region.

The positions of the cannula, diaphragm, heart, lungs, and liver wereverified by CT scans. In situ hybridization confirmed the gamma camerabiodistribution data. Cells containing human DNA were foundpredominantly in the liver.

Example 8 MSC Administered in Either Saline Alone, RPMI-1640 Alone, orSaline Containing 5% Human Serum Albumin (Without Orosomucoid or ASO)Migrate to the Lungs and Kidneys

MSC were obtained from BioWhittaker, (Poietics Division, cryopreservedPT-2501 >750000 cells per ampoule) and labeled with ¹¹¹In. As in Example6, the MSC were labeled, washed, and injected in saline alone, RPMI-1640medium (GIBCO BRL, Grand Island, N.Y.) and saline containing 5% humanserum albumin (HSA).

Saline alone.-1.14×10⁶ ¹¹¹In-labeled, MSC were administered i.v. in 0.20ml of saline alone. MSC were administered via an implanted DaVinciMicroport Vascular system cannula in the external jugular vein of atwo-month old, female NOD-SCID mouse.

Mice were imaged and the biodistribution of the radiolabeled MSCmonitored as in Example 1. Gamma camera monitoring initiallypost-infusion showed that the radiolabeled MSC localized to the regionof the lungs. Region of interest analysis of the initial images revealedthat 95% of the injected radioactivity localized to the lungs. At 1 hr,87% and 4% localized to the lungs and kidneys respectively; at 24 hr,61% and 13% localized to lungs and kidneys respectively; and at 48 hr,59% and 14% localized to the lungs and kidneys, respectively.

The positions of the cannula, diaphragm, heart, lungs, and liver wereverified by CT scans and a ⁵⁷Co-Spot Marker is used to mark anatomicpositions (nose, tail, cannula, etc.)

.RPMI-1640 alone. 1.14×10⁶ ¹¹¹In-labeled, MSC were administered i.v. in0.20 ml of RPMI-1640 alone.-MSC were administered via an implantedDaVinci Microport Vascular system cannula in the external jugular veinof a two-month old, female NOD-SCID mouse.

Mice were imaged and the biodistribution of the radiolabeled MSCmonitored as in Example 1. Gamma camera monitoring initiallypost-infusion showed that the radiolabeled MSC localized to the regionof the lungs. Region of interest analysis of the initial images revealedthat 95% of the injected radioactivity localized to the lungs. At 1hr,-74% and 7% localized to the lungs and kidneys, respectively, and at24 hr, 69% and 9% localized to lungs and kidneys respectively.

The positions of the cannula, diaphragm, heart, lungs, and liver wereverified by CT scans and a ⁵⁷Co-Spot Marker is used to mark anatomicpositions (nose, tail, cannula, etc.)

Saline containing 5% human serum albumin (HSA). 1.14×10⁶ ¹¹¹In-labeled,MSC were administered i.v. in 0.20 ml of saline containing 5% HSA. MSCwere administered via an implanted DaVinci Microport Vascular systemcannula in the external jugular vein of a two-month old, female NOD-SCIDmouse.

Mice were imaged and the biodistribution of the radiolabeled MSCmonitored as in Example 1. Gamma camera monitoring initiallypost-infusion showed that the radiolabeled MSC localized to the regionof the lungs. Region of interest analysis of the initial images revealedthat 94% of the injected radioactivity localized to the lungs. At 1 h;87% and 2% localized to the lungs and kidneys respectively; at 24 hr,59% and 11% localized to lungs and kidneys respectively; and at 48 hr,57% and 14% localized to the lungs and kidneys, respectively.

Results

The results of the experiments described above are summarized in Table1, below. TABLE 1 Summary of Results of Examples 1-8 % Infused StemCells/ % Infused Cells % Infused Cells % Infused Cells Cells ProteinBolus in Lungs in Liver/Spleen in Heart in Kidney HSC/ 78 ± 3% at 0 hrNo Protein 54 ± 10% at 12 hr HSC/ 74 ± 3% at 0 hr   Orosomucoid 74 ± 4%at 1.5 hr 63 ± 1% at 24 hr  HSC/ 77 ± 1% at 0 hr   ASO 75 ± 5% at 1.5 hr52 ± 1% at 24 hr  MSC/ 95% at 0 hr [considerable at 48 hr 4% at 1 hr NoProtein 87% at 1 hr Gao et al., Cells, 13% at 24 hr 61% at 24 hrTissues, Organs 14% at 48 hr [majority at 0 hr 169: 12-20 (2001)] Gao etal., Cells, Tissues, Organs 169: 12-20 (2001)] MSC/ 68 ± 7% at 0 hr  Orosomucoid 61 ± 3% at 1 hr   MSC/ 63 ± 5% at 0 hr   ASO 57 ± 7% at 1hr  

Example 9

The broad objectives of the following experiments was to determinewhether human LAK cell populations bind specifically to human hepatomacells via the ASGP receptor and, if so, how this cell recognition systemcould be manipulated for lymphocyte cell targeting. The generalexperimental approach uses similar sialo-asialo-containing plasmaproteins in an in vitro system mimicking contact with liver cellsbearing ASGP receptors, shown in FIG. 5.

Adherence of NK/LAK Activity To Human Minimal Deviation HepatomaMonolayers

Control cells (no IL-2 treatment) or LAK cells (IL-2-treated humanperipheral blood lymphocytes cultured 10U IL-2/ml for 3 days) wereadhered to a monolayer of HEP G2 cells for 2 hours at 4° C. Themonolayer was pretreated either with asialofetuin (ASF, 200 μg/ml) inmedia or with fetuin (F, control, 200 μg/ml)) in media. After theControl or LAK cells had been incubated on the monolayer, these cellswere then decanted, washed, and tested for cytotoxic capacity in a⁵¹Cr-release assay against the NK-resistant target, Raji. The E:T ratioswere 40:1, 20:1, 10:1, and 5:1; the standard error of the means isdisplayed; the E:T ratio is plotted as the LOG E:T. The results areshown graphically in FIG. 6.

CONCLUSION: LAK activity was reduced approximately 50% by incubatingthese cells on HEPG2 monolayers that had been treated with the control(fully sialated) protein, fetuin (which does not block the ASGPreceptor). LAK activity was not removed by incubating these cells onHEPG2 monolayer that had been preheated with asialofetuin (to block theASGP receptors). LAK or Control preparations that had been incubatedwith either fetuin or asialofetuin (at 200 μg/ml) for 2 h at 40C hadidentical activity to untreated LAK cell populations. These data supportthe notion that LAK cells bind to the hepatic ASGP receptor and thisbinding can be inhibited by blocking this receptor with asialofetuin.The extension of this finding is that hepatic sequestration of LAK cellsis at least in part due to the ASGP receptor and that the administrationof an asialoglycoconjugate, such as asialofetuin could prevent thisentrapment and alter LAK cell trafficking.

Adherence to HEPG2 (ASGP Receptor-Positive, “ASGPR+”) and CAKI-2 (ASGPReceptor-Negative, “ASGPR−”) at 23° C.

This experiment is the same as above, except that the adherence tomonolayers was performed at 23° C. and not 4° C., for 2 hours. Twomonolayers were used: HEPG2, an ASGPR+ cell line, and CAKI-2 (humanrenal cell carcinoma), an ASGPR− cell line. The effector cellpopulations that were used were: an untreated 3-day old LAK preparation(LAK) and the same population treated with Vibrio cholera neuraminidase(LAK/NS) (30 mU/1×10⁷ cells/200 μl). The neuraminidase-treatedpopulation was the asialopositive lymphocyte control. All cellpopulations regardless of treatment were greater than 90% viable at thetime of assay. Each type of effector population was incubated with mediaalone, 200 μg/ml ASF or F, as controls. All effectors were assayed onRAJI (LAK-sensitive target; NK-insensitive target) or K562(NK/LAK-sensitive target); the E:T ratios and the graphic presentationare the same above.

Results. The experiments above gave the following results (see FIGS.6-8). For the following discussion, activity on RAJI will be referred to“LAK” activity; activity on K562 will be referred to as “IL-2 activatedNK” activity. Some investigators support the idea that NK and LAKrecognize and kill targets (fresh and cultured tumor cells) using thesame target structures.

(1) Preincubation of effectors, either untreated (LAK) or treated(LAK/NS), with ASF or F, does not affect the ability of the effectors tokill either RAJI or K562 cells.

(2) Neuraminidase treatment enhances LAK activity on RAJI, but does notenhance IL-2 activated NK on K562 (see also FIGS. 11 & 12)

(3) Adherence to HEPG2 of IL-2 activated NK, with or withoutneuraminidase treatment, can be partially inhibited by ASF, but not by Fat 23° C. (FIGS. 7 & 8)

(4) Adherence to HEPG2 of LAK activity could not be inhibited witheither ASF or F at 23° C. (FIGS. 9 & 10)

(5) Adherence of LAK activity of the neuraminidase-treated population toHEPG2 could only marginally be inhibited by ASF and not F. (FIG. 10)

(6) Adherence to CAKI-2 of IL-2 activated NK or LAK activity could notbe inhibited by either ASF or F at 23° C. (FIGS. 7-10)

CONCLUSIONS: LAK activity (as determined on RAJI targets) and IL-2activated NK activity (as determined on K562 targets) display differentadherence characteristics to HEPG2, an ASGPR+ cell line. At 23° C. usingASF, LAK activity adherence to HEPG2 cannot be inhibited; whereas, IL-2activated NK adherence can be partially inhibited. At 4° C. virtuallyall LAK activity can be inhibited from adhering to the HEPG2 monolayerby ASF. Adherence to the CAKI-2 (ASGPR−) monolayers cannot be blocked byASF at 23° C.

These data suggest that adherence to the HEPG2 monolayer is in partmediated by the ASGP receptor and adherence to the CAKI-2 monolayer doesnot involve this receptor. A working hypothesis is that LAK/NK cellsbind to HEPG2 via at least two receptors or recognition structures: 1)the ASGPR, which binds an asialodeterminant on the LAK/NK population and2) the “LAK” or “NK” recognition structure for a target epitope. Thefirst should be inhibitable by ASF; the second should not be. Binding toCAKI-2 (ASGPR−) should not be inhibited by ASF and is due to a LAK or NKrecognition structure binding to the target epitope. This can be furthersupported by data derived from experiments (see below) in which 250μg/well of ASF or F were added to the ⁵¹Cr-release assay of LAKeffectors against the labeled target CAKI-2. Even at a concentration of1 mg/ml, ASF did not inhibit the ability of LAK to kill CAKI-2 target.

At 23° C. the ASGPR recycles and at 4° C. it does not, according toSchwartz. et al. Characterization of the ASGP receptor in a continuoushepatoma line. J. Biol. Chem 256: 88 78-(1981); Schwartz, A. L., et al.,Recycling of the ASGP receptor: biochemical and immunocytochemicalevidence. Phil. Trans. R. Soc. Lond. 300:229-235 (1982). The differencesseen in the ability to inhibit adherence may be explained by thetemperature dependence of ASGPR recycling and possibly of the LAKrecognition structure on the target. At 4° C. the LAK:target binding,both by the ASGPR and LAK recognition structure, may have the differentaffinity for ligand than at 23° C., or possibly at the increasedtemperature other adhesion molecules are capable of increasing theeffector:target interaction. That is, at 4° C. the only receptor onHEPG2 that binds LAK with any appreciable affinity is the ASGPR, andthis static receptor at this temperature can easily be inhibited by itsligand, ASF. At 23° C. more than the ASGPR binds the LAK cell to thetarget; the ASGPR is recycling in the presence of the ligand, ASF,leaving at least the LAK recognition structure for the target andpossibly other secondary adhesion molecules to “cement” the interaction.

These data also suggest that the LAK (as assayed on RAJI) and IL-2induced NK (as assayed on K562) cells have different affinity receptorsor different on/off rates for adherence to HEPG2.

Neuraminidase treatment, in theory, should have increased binding of theLAK cells to the HEPG2 monolayer due to the additional number ofasialodeterminants generated by this treatment, but did not. If thenumber of asialodeterminants was already sufficient to occupy themaximum number of ASGPR on HEPG2, increasing the number of thesedeterminants would not alter the end-effect. It is also possible thereis a specific asialodeterminant that is involved in the binding and thatgenerating more, but irrelevant determinants, will not increaseadherence. This suggests the interesting possibility that LAK and theIL-2 activated populations may differ in the ligands that participate inthis adherence to HEPG2.

LAK Cell Killing of Tumor Targets is not Blocked By ASF or F inPretreatment of Targets or When Added to the ⁵¹CR-Release Assay

Because asialodeterminants may play a role in both LAK-targetinteraction and LAK trafficking and liver adherence, it is important todetermine whether the use of asialoglycoprotein agents, in vivo, toalter trafficking patterns also inhibit cytotoxic activities, renderingsuch manipulations counterproductive. The preincubation of targets withthe addition of asialofetuin or fetuin to the assay, at 250 μg per well,does not block LAK killing of the tumor target, CAKI-2. The LAKpreparation was a standard 5-day preparation; however, these data havebeen replicated with 3-day LAK preparation. (% SPECIFIC RELEASE wasdetermined from quadruplicates whose raw counts per minute differed byless than 10 percent; the assay was a standard 4-hour incubation.) TABLE2 % ⁵¹Chromium Release from Caki-2 Targets AGENT ADDED TO ASSAY 40:120:1 10:1 5:1 Media 48 49 23 14 F 58 42 27 16 ASF 52 41 24 13Spontaneous release (media alone): 2183 cpm.Spontaneous release (fetuin alone): 2267 cpm.Spontaneous release (asialofetuin alone): 2147 cpmTotal release: 30,600 cpm.

Adherence of NK/LAK Activity After Cell Surface Modification byNeuraminidase or 2,3- and 2,6-Siayltransferases

Five-day LAK preparations (20 U/ml; 1 Dupont unit=44.5 BRMP units) grownin AIM-V (Gibco) were treated (according to the protocols in B. 1.2.3 &1.2.4) with 3 OmU Vibric Cholera neuramindase, 0.48 mU 2,3-or 10 mu2,6-silalyltransferase per 107 cells. Some of the effectors wereincubated in media, 10% PBS in RPMJ 1640 at 23° C. for 2 hours. 2×107effectors from the untreated LALK, neuramithdase-treated LAK, LAKtreated with 2,3 or 2,6 were suspended in 15 ml media and placed ontoeither HEPG2 (ASGPR+) or CAKI-2 (ASGPR−) monolayers at 23° C. for 2hours. The flasks were rocked every 15 minutes. The nonadherent cellsfrom these monolayers were decanted and assayed against K562 and RAJI,in addition to the unadhered controls. The results are presented inFIGS. 11-16. The E:T ratios used were 40, 20, 10, and 5 to 1.

Results. See Table 2, above, for Summary. Graphic presentation of thisdata in FIGS. 11-16).

(1) IL-2 activated NK (killing K562 targets) is not affected by any cellsurface modifications (FIGS. 11, 12 and 13, top 4 dotted lines);whereas, LAK activity (killing of RAJI) is significantly enhanced byneuraminidase treatment (FIGS. 12, 15 & 16), but not by 2,3-or2,6-sialyltransferase treaments FIGS. 15 & 16).

(2) No modification of LAK cell surfaces alters adherence to CAKI-2 ascompared to untreated LAK (assayed on RAJI) (FIG. 15). In contrast,neuraminidase treatment promotes adherence to CAKI-2 of IL-2 activatedNK activity (FIG. 14, bottom solid line; assayed on K562) as well as2,3-sialyltransferase treatment (FIG. 14, solid line aboveneuraminidase). Treatment with 2,6-sialyltransferase has no effect onthe adherence to CAKI-2 of either LAK or IL-2 activated NK.

(3) No modification of the cell surface dramatically modifies adherenceof LAK activity to HEPG2 (FIG. 15); however, 2,6-sialyltransferasetreatment significantly promotes adherence of IL-2 activated NK (FIG.13, bottom solid); and conversely, 2,3-sialyltransferase treatmentsignificantly prevents adherence of these cells to HEPG2 (FIG. 13, topsolid line).

CONCLUSIONS: IL-2 activated NK killing and LAK are affected differentlyby neuraminidase treatment.

IL-2 activated NK adherence to both HEPG2 were altered by cell surfacemodifications; LAK adherence was not affected by these modifications.This may be due to the amount of sialic acid that can be added to theLAK cell surface which could be determined by dose-response of 2,3- and2,6-sialyltransferases.

Adherence of IL-2 activated NK to HEPG2 at 23° C. could be partiallyinhibited by ASF (previously reported) and by adding sialic acid with2,3-sialyltransferase (while 2,6-sialyltransferase treatment promotedadherence).

It is necessary to determine whether adding higher concentrations of ASF(or another asialocompound, e.g., asialoGMI-sugar) as a means ofcompensating for ASGPR-recycling at 23° C. or even at 37° C. can preventadherence of IL-2 activated NK or LAK. Likewise, performingdose-response experiments with 2,3- and 2,6 sialyltransferase to achieveaddition of the maximum amount of sialic acid may allow the dissectionof the adherence mechanism because each enzyme adds to differentstructures: 2,3- to O-linked sugars linked to ser/thr and2,6-sialyltransferase to N-linked sugars linked to asn. Theseglycosyltranferases may be equally important in discriminating betweenthe populations responsible for IL-2 activated NK (killing of K562) andthose responsible for RAJI killing, LAK.

All publications, patents, patent applications, and other documentsmentioned in the specification are indicative of the level of thoseskilled in the art to which this invention pertains. All publications,patents, patent applications, and other documents are hereinincorporated herein by reference in their entirety for all purposes tothe same extent as if each individual publication, patent, patentapplication, or other document was specifically and individuallyindicated to be incorporated herein by reference in its entirety for allpurposes. Subheadings are included solely for ease of review of thedocument and are not intended to be a limitation on the contents of thedocument in any way.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A method for delivering a stem cell or lymphoid cell to a targettissue in a mammal comprising the steps of: (a) administering aglycoconjugate to a mammal; (b) administering the cell to the mammal. 2.The method of claim 1, wherein the cell is a hematopoietic stem cell. 3.The method of claim 2, wherein the stem cell is obtained from the bonemarrow, placenta, muscle, fat or an umbilical cord.
 4. The method ofclaim 1 wherein the lymphoid cell is selected from the group consistingof a natural killer (NK) cell, a lymphokine-activated killer (LAK) cell,a tumor-infiltrating lymphocyte (TIL), a cytotoxic lymphocyte (CTL), andmixtures thereof.
 5. The method of claim 1, wherein the glycoconjugateis represented by the general formula P-(S)x-Gal, wherein P is a peptideresidue of a human serum glycoprotein and S is a sugar residue of ahuman serum glycoprotein; x is an integer from 1 to 100 and Gal isgalactose residue.
 6. The method of claim 1, wherein the glycoconjugateis selected from the group consisting of an orosomucoid and anasialoorosomucoid.
 7. The method of claim 1, wherein the target tissueis a tissue of an organ selected from the group consisting of the heart,the liver, the lungs, and the kidneys.
 8. The method of claim 1, whereinthe glycoconjugate is administered to the mammal prior to the cell. 9.The method of claim 1, wherein the glycoconjugate and the cell areadministered intravenously to the mammal.
 10. A method for targeting ahematopoietic stem cell to the heart of a mammal comprising the stepsof: (a) administering an asialo-orosomucoid to the mammal; and (b)administering the cell to the mammal.
 11. The method of claim 10,wherein the cell is administered after the step of administering theasialo-orosomucoid.
 12. The method of claim 10, wherein theasialo-orosomucoid is administered via a vessel proximal to the heart.13. The method of claim 12 wherein the asialo-orosomucoid isadministered via a jugular vein.
 14. The method of claim 10 wherein theheart of a mammal has suffered ischemic injury prior to administeringthe asialo-orosomucoid.
 15. A method for targeting a mesenchymal stemcell to the heart of a mammal comprising the steps of: (a) administeringan orosomucoid to the mammal; and (b) administering the cell to themammal.
 16. The method of claim 15, wherein the orosomucoid isadministered via a vessel proximal to the heart.
 17. The method of claim16 wherein the orosomucoid is administered via a jugular vein.
 18. Themethod of claim 15 wherein the heart of a mammal has suffered ischemicinjury prior to administering the orosomucoid.
 19. The method of claim15, wherein the cell is administered after the step of administering theorosomucoid.
 20. A method for targeting a hematopoietic stem cell to theliver of a mammal comprising the steps of: (a) administering anorosomucoid to the mammal; and (b) administering the cell to the mammal.21. The method of claim 20, wherein the cell is administered after thestep of administering the orosomucoid.
 22. A method for targeting amesenchymal stem cell to the liver of a mammal comprising the steps of:(a) administering an asialoorosomucoid to the mammal; arid (b)administering the cell to the mammal.
 23. The method of claim 22,wherein the cell is administered after the step of administering theorosomucoid.
 24. A method for targeting a gene of interest to a tissuein a mammal, wherein said ene of interest comprises a transgene, saidmethod comprising the steps of: (1) introducing a cell comprising thegene of interest to the mammal; and (2) administering a glycoconjugate.25. The method of claim 24, wherein the cell is a hematopoietic stemcell.
 26. The method of claim 24, wherein the cell is a lymphoid cell.27. The method of claim 26, wherein the stem cell is obtained from thebone marrow, peripheral circulation or an umbilical cord.
 28. The methodof claim 24, wherein the glycoconjugate is selected from the groupconsisting of an orosomucoid and an asialoorosomucoid.
 29. A method fortreating a disease characterized by tissue damage in a mammal comprisingthe steps of: (1) administering a stem cell to the mammal; and (2)administering a glycoconjugate to the mammal.
 30. The method of claim29, wherein the stem cell is obtained from the hone marrow, peripheralcirculation or an umbilical cord.
 31. The method of claim 29, whereinthe glycoconjugate is selected from the group consisting of anorosomucoid and an asialoorosomucoid.
 32. The method of claim 29,wherein the disease is selected from the group consisting of a heartdisease, a lung disease, a liver disease a neurological disease and akidney disease.
 33. The method of claim 29, wherein the disease isselected from the group consisting of myocardial infarction, emphysema,cystic fibrosis, hepatitis, stroke, nephritis and microalbuminuria. 34.A pharmaceutical composition comprising a lymphoid cell or a stem celland a glycoconjugate.
 35. The pharmaceutical composition of claim 34,wherein the glycoconjugate is selected from the group consisting of anorosomucoid and an asialoorosomucoid.
 36. The pharmaceutical compositionof claim 34, wherein the cell is a stem cell.
 37. The pharmaceuticalcomposition of claim 34, wherein the cell is a lymphoid cell.
 38. Anarticle of manufacture, comprising packaging material and apharmaceutical composition contained within the packaging material,wherein the pharmaceutical composition comprises a glycoconjugate thatis therapeutically effective for targeting a cell to a desired organ,and wherein the packaging material comprises a label which indicatesthat the pharmaceutical composition can be used for targeting a cell toa desired organ.
 39. The article of manufacture of claim 38, furthercomprising additional reagents for making cell suspensions to beadministered to a mammal and printed instructions, for use in targetingcells.
 40. The article of manufacture of claim 39 further comprising aquantity of stem cells suitable for targeting of such cells in a mammal.41. The article of manufacture of claim 38, wherein the glycoconjugateis selected from the group consisting of an orosomucoid and anasialoorosomucoid.
 42. The article of manufacture of claim 40, whereinthe cell is a hematopoietic stem cell.
 43. A method to improve theefficiency of an adoptive immunotherapy using a lymphoid cell comprisingmodification of sialoglycoprotein determinants on the lymphoid cellsurface.
 44. The method of claim 43 wherein the modification comprisesremoval of sialic to generate new asialoglycoprotein determininants. 45.The method of claim 44 wherein the modification comprises removal ofsialic acid by an enzyme.
 46. The method of claim 45 wherein themodification comprises removal of sialic acid by a neuraminidase. 47.The method of claim 43 wherein the modification comprises addition ofsialic acid by an enzyme.
 48. The method of claim 43 wherein theadoptive immunotherapy is for a liver metastasis or a primary livertumor. regional administration to the liver of activated lymphocytes.49. The method of claim 6 wherein the glycoconjugate is administered viaa vessel proximal to the organ wherein the target tissue is located. 50.The method of claim 6 wherein the organ is the liver and theglycoconjugate is administered viavia the hepatic artery or portal veinor peripheral vein